Recombinase Polymerase Amplification

ABSTRACT

This disclosure describes related novel methods for Recombinase-Polymerase Amplification (RPA) of a target DNA that exploit the properties of recombinase and related proteins, to invade double-stranded DNA with single stranded homologous DNA permitting sequence specific priming of DNA polymerase reactions. The disclosed methods have the advantage of not requiring thermocycling or thermophilic enzymes, thus offering easy and affordable implementation and portability relative to other amplification methods. Further disclosed are conditions to enable real-time monitoring of RPA reactions, methods to regulate RPA reactions using light and otherwise, methods to determine the nature of amplified species without a need for gel electrophoresis, methods to improve and optimize signal to noise ratios in RPA reactions, methods to optimize oligonucleotide primer function, methods to control carry-over contamination, and methods to employ sequence-specific third ‘specificity’ probes. Further described are novel properties and approaches for use of probes monitored by light in dynamic recombination environments.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/212,361, filed Aug. 18, 2011, now U.S. Pat. No. 8,426,134, which is acontinuation of U.S. application Ser. No. 12/660,117, filed Feb. 19,2010, now U.S. Pat. No. 7,666,598, which is a divisional of U.S.application Ser. No. 11/628,179, filed Aug. 30, 2007, now U.S. Pat. No.7,666,598, which is a National Stage of International Application No.PCT/IB2005/001560, filed Apr. 11, 2005, which claims the benefit ofpriority to Provisional Application No. 60/576,148, filed Jun. 1, 2004,Provisional Application No. 60/576,162, filed Jun. 1, 2004, ProvisionalApplication No. 60/622,291, filed Oct. 26, 2004, Provisional ApplicationNo. 60/632,746, filed Dec. 2, 2004, and is also a continuation-in-partof U.S. application Ser. No. 10/931,916, filed Sep. 1, 2004, now U.S.Pat. No. 7,399,590, which claims the benefit of priority to ProvisionalApplication No. 60/553,999, filed Mar. 16, 2004. U.S. application Ser.No. 10/931,916 is also a continuation-in-part of U.S. application Ser.No. 10/371,641, filed Feb. 21, 2003, now U.S. Pat. No. 7,270,981, whichclaims the benefit of priority to Provisional Application No.60/358,563, filed Feb. 21, 2002. All of the foregoing applications areincorporated by reference herein.

BACKGROUND OF THE INVENTION

The ability to amplify DNA lies at the heart of modern biological andmedical research. This is because most molecular biology techniques relyon samples containing many identical molecules to increase thesensitivity of an assay or to prepare enough material for furtherprocessing. Among the various nucleic acid amplification techniques,polymerase chain reaction (PCR) is the most common because of itssensitivity and efficiency at amplifying short nucleic acid sequences.

While PCR is of great utility, it is also limited in a number of ways.The first limitation of PCR is that it relies on multiple cycles ofthermal melting (denaturing) at high temperatures followed byhybridization and elongation at a reduced temperature. To maximizeefficiency and to minimize noise, complex temperature control ofmultiple reactions is required. This necessitates the use of athermocycler controllable rapid heating/cooling block made with exoticmaterial (e.g., gold plated silver blocks), or a robotic mechanism tomove samples between temperature-controlled zones. Because of thehigh-temperature required to melt DNA in physiological salt conditions,PCR technology requires either the addition of fresh polymerase percycle or the use of thermostable polymerases. The approach of addingfresh polymerase has not been automated and is thus labor intensive andprone to errors (e.g., contamination, dropped tubes, labeling errors).Furthermore, the need to add enzymes and to mix each reactionindividually presents serious drawbacks that have limited adaptation ofenzyme-addition PCR methods to the small scale.

Compared to methods involving the addition of fresh polymerase, the useof thermostable polymerases in PCR is the most widely practiced. Thisapproach suffers from the fact that thermostable polymerases are foundin a limited number of organisms, and the replication mechanisms used bythermophilic organisms are poorly understood. The available repertoireof thermostable polymerases is limited to single polypeptide polymeraseenzymes involved in DNA repair, and/or lagging strand synthesis. DNArepair and/or lagging strand polymerases are poor choices for DNAamplification because they exhibit poor processivity (distributivesynthesis). In part as a consequence of using repair and/or laggingstrand polymerases (e.g. Taq, Pfu, Vent polymerases), and due to theformation of inhibitory secondary or tertiary nucleic acid structuresfollowing thermal melting, current PCR protocols do not readily amplifysequences longer than several thousands of base pairs. Reliablesynthesis (and amplification) of longer templates will rely onpolymerases and auxiliary enzymatic complexes collectively exhibitingmuch higher levels of processivity, strand displacement, and secondarystructure resolution, as well as limiting the formation of inhibitoryhigher order nucleic acid structures that may form on coolingheat-denatured DNA.

A second limitation of PCR is that it relies on solution hybridizationbetween oligonucleotides (PCR primers) and denatured template DNA (i.e.,the DNA to be amplified) in an aqueous environment. To be effective, PCRreactions are performed in a short time because the thermostablepolymerases have a rapidly declining activity at PCR temperatures.Further, for effective hybridization in a short time, a feature criticalto rapid turnaround, it is necessary to perform PCR in an environmentwith high concentrations of oligonucleotides. The high oligonucleotideconcentration also ensures rapid interaction of target sequences withthe oligonucleotides in competition with the heat-denaturedcomplementary strand still present in solution. High oligonucleotideprimer concentrations can cause problems, particularly when the copynumber of the target sequence is low and present in a complex mixture ofDNA molecules. This would be the case, for example, in a PCR of a genometo determine the genetic polymorphism in one locus.

One problem with using high oligonucleotide concentrations is that itenhances the degree of false priming at only partly matched sequences inthe complex DNA mixture. False priming refers to the hybridization of aprimer to a template DNA in PCR even when the primer sequence is notcompletely complementary to the template nucleic acid, which can lead tonon-specific amplification of nucleic acids. Noise, due to falsepriming, increases with the oligonucleotide concentration and thecomplexity of total starting DNA. In addition, the possibility of falsepriming increases as the copy number of target sequences decreases.Where the conditions for false priming are favorable (i.e., higholigonucleotide concentration, high complexity, low copy number), errantamplified sequences can become a dominant reaction product. Consequentlyit can be difficult to identify conditions, and oligonucleotides, forclean amplification of target sequences from a sample DNA without anexcess of false priming background. Thus a further disadvantage of usingPCR is the limited success at cleanly amplifying rare target DNAs fromcomplex sequences mixtures.

One solution to the problems of specificity and template-melting problemincurred by PCR is to employ methods that rely on the biologicalproperties of the bacterial RecA recombinase protein, or its prokaryoticand eukaryotic relatives. These proteins coat single-stranded DNA(ssDNA) to form filaments, which then scan double-stranded DNA (dsDNA)for regions of sequence homology. When homologous sequences are located,the nucleoprotein filament strand invades the dsDNA creating a shorthybrid and a displaced strand bubble known as a D-loop. The free 3′-endof the filament strand in the D-loop can be extended by DNA polymerasesto synthesize a new complementary strand. The complementary stranddisplaces the originally paired strand as it elongates. By utilizingpairs of oligonucleotides in a manner similar to that used in PCR itshould be possible to amplify target DNA sequences in an analogousfashion but without any requirement for thermal melting (thermocycling).This has the advantage both of allowing the use of heat labilepolymerases previously unusable in PCR, and increasing the fidelity andsensitivity by template scanning and strand invasion instead ofhybridization.

Although the use of RecA and its homologues for in vitro amplificationof nucleic acids has been previously described (U.S. Pat. No. 5,223,414to Zarling et al., referred to herein as “Zarling”), the method andresults are limited. Zarling's method has critical failings that limitits ability to achieve exponential amplification of double-stranded DNA.The failure of the Zarling method to achieve exponential amplificationmay be due to its specification for the use of ATPγS rather than ATP.The Zarling method urges the use of ATPγS, instead of ATP, in theassembly of RecA nucleoprotein filaments because it results in a morestable RecA/ssDNA filament structure. Normally, filaments are assembledin a 5′ to 3′ direction and will spontaneously disassemble in the same5′ to 3′ direction as RecA hydrolyzes ATP. This process is dynamic inthat assembly and disassembly occurs at the same time and the amount ofassembled filaments is at equilibrium. If the non-hydrolyzable ATPanalog, ATPγS, is used, hydrolysis of the ATPγS and the 5′ to 3′disassembly of the filaments are inhibited. The great stability ofRecA/ATPγS filaments, both before and after strand exchange, whilehelpful in the method of targeting (i.e., the Zarling method) isdetrimental and unpractical for DNA amplification.

In the Zarling method, RecA protein involved in strand invasion willremain associated with the double-stranded portion of the exchangedmaterial after strand exchange. This interaction occurs because thenewly formed duplex is bound in the high-affinity site of RecA. Thedisplaced strand occupies a different low-affinity site, unless it isbound to another single-stranded DNA binding protein (SSB), such as E.coli SSB. If ATP had been utilized to generate the exchange structure,spontaneous 5′ to 3′ disassembly might occur, although the exchangecomplex can be quite stable and may require additional factors tostimulate ATP-dependent disassembly. Regardless of whether spontaneousor stimulated, in the presence of ATPγS, 5′ to 3′ disassembly of theRecA filament is inhibited (Paulus, B. F. and Bryant, F. R. (1997).Biochemistry 36, 7832-8; Rosselli, W. and Stasiak, A. (1990). J Mol Biol216, 335-52; Shan, Q. et al., (1997). J Mol Biol 265, 519-40).

These RecA/dsDNA complexes are precisely the sites targeted by theRecA/ssDNA primer complexes used to initiate subsequent rounds ofinvasion and synthesis. Indeed, with the RecA bound, the intermediatemay not be accessible to polymerase, and certainly the dsDNAs can nolonger be invaded by RecA/ssDNA primer complexes and are therefore notamplifiable from this point. Further synthesis from these templatesmight occur if initiated at the other end of the template, which is freeof RecA, and this might eventually lead to physical displacement of thebound RecA. It is not clear, however, whether many polymerases candisplace RecA in this manner. Moreover, the initiation site for thatsynthetic round will now be ‘blocked’ instead. In such a situation,amplification is only linear with time, and will predominately generatesingle-stranded DNA amplification products.

Thus, the described Zarling method, at best, is likely to generatelittle more than small quantities of ssDNA copies from each template.The linear amplification potentially given by the Zarling method willonly occur in the presence of SSB, since the displaced strand willcontinue to bind to the second interaction site on RecA, andsingle-stranded DNA will not be released (Mazin, A. V. andKowalczykowski, S. C. (1998). EMBO J 17, 1161-8). This probably explainswhy the Zarling method observed additional faster-migrating fragmentswhen they included SSB. These additional fragments were most likelydisplaced single-stranded fragments. Hence, in the Zarling method onlylinear amplification of single-stranded DNA will occur at best. Thereis, therefore, a need in the art for an improved recombinase-dependentDNA amplification method.

This invention utilizes two new amplification strategies that avoid anyrequirement for thermal melting of DNA or thermostable components. Thesestrategies also overcome the inefficiencies of the Zarling method. Aswith the Zarling strategy, these methods rely on the biologicalproperties of the bacterial RecA protein, or its prokaryotic andeukaryotic relatives, in particular, the phage T4 uvsX protein. However,in contrast to the Zarling method, these methods are devised to achieveexponential amplification of dsDNA. They achieve this by permittingrapid regeneration of targetable sequences in the target nucleic acid inthe presence of dynamic recombinase/DNA filaments, rather than ATPγSloaded non-dynamic filaments, and in an environment that concomitantlysucceeds in maintaining high recombination activity. Furthermore, andcritically, while the concept of elongating from recombinationintermediates has been visited earlier in concept, and limited practice,both in the Zarling approach, and also in the Alberts laboratory(Formosa and Alberts, 1996; Morrical and Alberts, 1990; Morrical, Wong,and Alberts 1991) and elsewhere (Salinas, Jiang, and Kodadek, 1995;Morel, Cherney, Ehrlich, and Cassuto, 1997; International patentapplication WO 02/086167, Benkovic and Salinas), none of thedescriptions to date teaches a practical method to allow exquisitelyspecific, sensitive exponential DNA amplification with a capacity foramplification up to 10 to the power of 12 fold. This is becauseestablishing this necessary environment which supports highrecombinase/filament activity, but in the presence of large quantitiesof the necessary single-stranded DNA binding proteins in an in vitroenvironment has proved extremely challenging, and this environment isentirely dependant on a strict combination of components. This includes,most critically and unexpectedly, very specific crowding agents whichalter the behaviour of the in vitro system in a remarkable, andessentially unpredictable way. This remarkable and largely unpredictablealteration of system behaviour with specific volume-occupying agentspresumably reflects their capacity to engender fractal-like kinetics,phase separation effects, or other additional properties on thebiochemical system. By identifying such precise conditions to enablerapid and highly geometric DNA amplification, as well as conditions fordriving high persistent and dynamic recombination activity in vitro forother uses, this invention enables a new generation of in vitromolecular techniques. We refer to the described amplification methodperformed under these enabling conditions as Recombinase PolymeraseAmplification (RPA). We envision herein yet further methods based uponthis high activity, persistent, yet dynamic recombination environment,which will likely become practiced in due course. This invention enablesthis new generation of approaches, and should be contrasted to thecurrent circumstance in which, despite decades of research, no otherwidely used application of recombinases for in vitro technology hasappeared apart from a very limited number dependant on the use of ATPγS.

In this invention we go further and demonstrate that RPA reactions canbe fully integrated with dynamic detection of reaction products. Thisvalidates that RPA reactions achieve two general criteria for real-timeanalysis. First a biochemical sensor, such as sensing dye like SYBRgreen or ‘third’ probe, is compatible with the RPA reaction environment.Such compatibility is not a trivial assumption because RPA employssaturating quantities of DNA binding proteins, which might interferewith dye or probe binding behaviour. Conversely the binding of dyes orprobes to nucleic acids might have interfered with the activity of theDNA binding proteins. Secondly to be employed in real-time quantitativeapplications RPA would need to demonstrate exponential DNA amplificationof target DNA over a significant range of starting template quantities,and be able to maintain exponential amplification up to concentrationseasily within the detection range of the overall sensor system.

Also in this invention we disclose approaches to control, andpotentially synchronise aspects of, RPA reactions. In currentconfigurations of RPA there is no temporal separation between the DNAtargeting and DNA synthesis phases. For RPA, it is difficult to ensurethat all reactions in RPA are initiated at exactly the same momentunless a rate-limiting reagent is supplied to all samplessimultaneously, or the reactions are assembled at a non-permissivetemperature. We suggest approaches by which RPA reactions may beinitiated, and individual ‘rounds’ of priming activity may be regulated,by limiting invasion to well-spaced short bursts. Such approaches tolimit recombinase activity to short limited bursts could improveamplification. One way to control DNA invasion in RPA may be byregulating the concentration of free ATP. In the absence of sufficientATP, or an excess of ADP, recombinase/DNA filaments disassemble andrecombination halts. Caged ATP does not support recA loading, butsubsequently uncaged material does [Butler B C, Hanchett R H, RafailovH, MacDonald G (2002) Investigating Structural Changes Induced ByNucleotide Binding to RecA Using Difference FTIR. Biophys J 82(4):2198-2210]. Thus the use of caged ATP analogues in RPA reactions, whichcan be deprotected in pulses by light thus permitting bursts ofrecombinase activity, should be an effective means to control theinvasion phase of an RPA reaction. Alternatively ATP concentration couldbe cyclically controlled by alternative methods such as periodicaddition of ATP to the reaction from an external source, or byestablishing a biochemical oscillator capable of generating periodicincreases of ATP in the reaction.

In this invention we extend the knowledge of how to attain idealrecombinase/ssDNA loading by virtue of 5′ sequence design, and widen therepertoire of contexts in which this key stable dynamic recombinationenvironment can be employed in addition to DNA amplification reactions.We describe how this unique composition may be used to replace classicalhybridisation steps in any process that otherwise would require thermalor chemical melting, or other duplex targeting approach, in a variety ofmolecular applications. In particular the use of stable dynamicrecombination environments in the presence of synthetic oligonucleotideswill be useful in combination with other enzyme systems than thepolymerase systems previously described, due to the lack of need forthermal or chemical melting, and the concomitant capacity to employ awider range of enzymes and avoid thermal cycling equipment.

SUMMARY OF THE INVENTION

The invention provides a method of DNA amplification, termed RPA, whichcomprises the following steps. First, a recombinase agent is contactedwith a first and a second nucleic acid primer to form a first and asecond nucleoprotein primer. Second, the first and second nucleoproteinprimers are contacted to a double stranded target sequence to form afirst double stranded structure at a first portion of said first strandand form a double stranded structure at a second portion of said secondstrand so the 3′ ends of said first nucleic acid primer and said secondnucleic acid primer are oriented towards each other on a given templateDNA molecule. Third, the 3′ end of said first and second nucleoproteinprimers are extended by DNA polymerases to generate first and seconddouble stranded nucleic acids, and first and second displaced strands ofnucleic acid. Finally, the second and third steps are repeated until adesired degree of amplification is reached.

The invention also provides for a method of nested RPAs. In a nestedRPA, a first region of nucleic acid is amplified by RPA to form a firstamplified region. Then a second region of nucleic acid that iscompletely within the first amplified region is amplified using RPA toform a second amplified region. This process may be repeated as often asnecessary. For example, a third region of nucleic acid, which iscompletely within the second region, may be amplified from the secondamplified region by RPA. In addition to the one, two and three rounds ofRPA discussed above, the invention contemplates at least 4, andpreferably at least 5 rounds of nested RPAs also.

The invention also provides for methods of detecting a genotype usingRPA. This method is useful for genotyping, for detecting a normal ordiseased condition, a predisposition, or a lack of a disposition for adiseased condition. Further, RPA can be used for detecting the presenceof a genome, such as for example, a genome of a pathogen. In this use,the method is useful for diagnosis and detection.

The invention also details the nature and concentrations ofrecombinases, single-stranded binding proteins, polymerases, andnucleotides necessary to establish an effective amplification reaction.The invention further provides detailed enablement on the nature of thetarget DNA, the length, and composition of targeting oligonucleotides,and the inter-oligonucleotide length optimal for amplification undervarious conditions. The invention provides for the inclusion ofadditional components, or the use of modified components, thatcontribute to establishing a recombination-polymerase amplificationsystem that is sensitive, robust, and with optimal signal-to-noiseproperties. In particular the use of more than one species ofrecombinase is demonstrated, and the utility of engineered and modifiedanalogues of the recombinases E. coli recA and T4 bacteriophage uvsX, ofpolymerases including the E. coli DNA polymerase I Klenow fragment, Bstpolymerase, Phi-29 polymerase, Bacillus subtilis Pol I (Bsu), as well assingle-stranded DNA binding proteins from E. coli and T4 (the gp32protein) is detailed.

The utility of forms of gp32 with altered cooperativity and/or strandassimilation properties is demonstrated. Also shown is the use of T4uvsY protein, and most particularly of molecular crowding agentsespecially PEG compound (also known as Carbowax 20M), to aid inestablishing an optimal reaction environment. Further the presentinvention details effects and the possible use of other enzymes involvedin DNA metabolism including toposiomerases, helicases and nucleases, inorder to improve the amplification behaviour. The present invention alsoincludes the use of optimised conditions for repeated invasion/extensionof a primer targeted to a supercoiled or linear template to generate alinear amplification, and the use of this method for DNA sequencing. Thepresent invention also describes the use of a recombinase in detectionof a specific amplified product of a reaction by directingoligonucleotides labeled in some manner to the specific product speciesand measuring a change in the appearance or property of the reactants asa consequence.

This invention also provides data and approaches to improve theimplementation of the RPA method, notably for diagnostic applications.Careful design of oligonucleotide length, base composition, and use ofmodified backbone sugar residues underpin strategies for highsensitivity and specificity tests. We also disclose approaches tocombine oligonucleotides with distinct activities as nucleoproteinfilaments to improve signal-to-noise ratios. Also disclosed are methodsof product detection that obviate gel electrophoresis, in some casesemploying ‘third’ specific oligonucleotides. We disclose theconstitution of an active lyophilizate that can be stored at ambienttemperature for at least 10 days and retain amplification activity whenreconstituted with buffered sample only.

This invention also discloses enabling data to permit the use of the RPAmethod in quantitative real-time applications. We show that appropriatedilutions of SYBR green or SYBR gold fluorescent nucleic acid bindingdyes are compatible with RPA reactions and permit the monitoring of theaccumulation of products. Products continue to accumulate in anapparently exponential fashion for sufficient time to permitquantification after threshold detection levels are achieved. We showexperimentally that using this approach RPA is quantitative over atleast four or five orders of magnitude of starting template quantity.Simultaneous initiation of many parallel reactions is achieved byestablishing reaction mixes on ice then simultaneously shifting thesamples to reaction temperatures (33-39° C.). Alternatively parallel RPAreactions might be simultaneously initiated by other means, such aslight-driven uncaging of ATP, or of caged oligonucleotide primers. Wedetail other product-specific real-time monitoring approaches that maybe compatible with the RPA system. We also describe the overallcomposition of a real-time RPA device composed of low power solid statecomponents which could enable cheap portable implementation in bothlaboratory and non-laboratory contexts.

This invention also discloses method to control RPA reactions, achievedby controlling the presence of the necessary supporting nucleosidetriphosphate cofactors such as ATP, to limited periods during thereaction. If chemically caged nucleotide triphosphates are used, pulsesof free ATP may be generated by illumination with a defined burst oflight corresponding to the uncaging wavelength of the photoprotectinggroup. The released ATP will then allow the binding of recombinaseproteins to single-stranded DNA (ssDNA), and the subsequent homologysearching and strand exchange activity of the recombinase-ssDNAcomplexes. Alternatively, ATP may be periodically added to the reaction.Over time, the concentration of ATP will decrease as a consequence ofhydrolysis, either recombinase hydrolysis or hydrolysis by otherreaction components specifically added to hydrolyse excess ATP, and ADP.Consequently after a period of time defined by the decreases in ATPconcentration and/or an increase in the ADP concentration, recombinasemolecules will cease to function and dissociate from DNA. Subsequentpulses of light at the uncaging wavelength can be delivered to releasefresh ATP, or fresh ATP can be added to re-initiate recombinaseactivity. In this manner a series of controlled periods of homologysearching and priming are enabled so that initiation of elongation canbe phased.

As a second method to control the invasion phase of RPA reactions, wedescribe an approach to separate the activity driven by one primer fromthe other. This method utilises recombinase-mediated invasion at oneprimer target site and the completion of synthesis from that primer.This will generate a single-stranded displaced DNA, which can then beused as a template for a second facing primer that is modified and thusunable to support recombinase-mediated initiation of synthesis. Thiswill possibly avoid conflicts arising through collision of polymerases.

This invention also discloses approaches to assess the polymorphicnature of amplified products without a need for size fractionation.Amplification reaction products are permitted to form duplex hybridswith immobilized probes of either initially single or double strandedcharacter by the action of recombinases and/or single-stranded DNAbinding proteins and other accessory molecules. Methods to destabilizeimperfect hybrids formed between products and probes are described,occurring in a dynamic environment of recombinase action, and supportiveof the activity of a wide variety of additional enzymatic components.Productive hybrids are detected by one of many standard approaches usedto reveal the presence of absence of a molecular interaction.

This invention also describes the combination of the determined in vitroconditions which support a stable persistent dynamic recombinationenvironment with other enzymatic activities, thus permitting strandinvasion and pairing between ssDNAs and duplexes to occur continuouslyand in the presence of other metabolic enzymes, especially thosenon-thermophilic enzymes that would be necessitated in conventionalapproaches, as well as other processes that are not equivalent, orattainable, in a system employing thermal or chemical melting. We alsodescribe findings that improve the design of oligonucleotides to permithigh activity in stable dynamic recombination systems by virtue ofincluding optimised sequences within, and particularly at the 5′ end ofdesired sequences.

Other embodiments, objects, aspects, features, and advantages of theinvention will be apparent from the accompanying description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a schematic representation of RecA/Primer Loading. Priorto the addition of template DNA and/or Polymerase, RecA and SSB-willcompete for binding to single-stranded oligonucleotide primers. In thepresence of a RecR and RecO, RecA is selectively stabilized onto thesingle-stranded primers forming RecA nucleoprotein filaments in acomplex with RecO and RecR. This complex is competent to invadedouble-stranded DNA to form a D-loop at sites homologous to theoligonucleotide primers. Alternatively, RecA, RecO and RecR can bepre-loaded onto oligonucleotide primers prior to the introduction of SSBto the reaction mixture.

FIGS. 2A-2B depict a schematic of succeeding steps, shown in panels (A)and (B), of Leading Strand Recombinase Polymerase Amplification (lsRPA).RecA/primer nucleoprotein filaments invade double stranded template DNApreferentially associating with homologous target sites. As D-loops areformed and synthesis proceeds displaced single stranded DNA becomescoated with SSB. RecA release from double-stranded DNA can occur via ATPhydrolysis in a 5′-3′ direction or as a result of helicase/resolvase orpolymerase activity. As synthesis continues, polymerases encounter SSBbound, displaced, single-stranded template. Double-stranded target siteare re-invaded by RecA/primer nucleoprotein filaments. Subsequent roundsof lsRPA proceed from re-invaded sites.

FIGS. 3A-3D depict a schematic of succeeding steps, shown in panels (A),(B), (C), and (D), of Leading and Lagging Strand Recombinase PolymeraseAmplification. First, the primosome loads onto the D-loop formed by RecAnucleoprotein filament invasion The primosome synthesizes a stretch ofRNA primer. Finally. primosome recruits the clamp loader, which recruitsboth the sliding clamp dimer and the asymmetric DNA polymerase core.Synthesis occurs simultaneously in both the leading and laggingdirections. Eventually lagging strand synthesis stops and the laggingstrand clamp is unloaded. Synthesis of the leading strand continuesuntil a new site of lagging strand synthesis is formed. While leadingstrand synthesis continues a new site of lagging strand synthesis isformed. Lagging strand synthesis continues back to the previous Okazakifragment where the lagging strand clamp is unloaded. DNA Polymerase Iremoves the RNA primer, and fills in the gap while DNA ligase connectsthe two Okazaki fragments forming a continuous lagging strand.

FIG. 4 depicts an example of nested primers chosen for nested RPA.

FIG. 5 depicts examples of suitable double stranded template nucleicacids.

FIGS. 6A-6B depict in panels (a) and (b) the various orientations of theRPA primer pairs in hybridization with the target nucleic acid.

FIGS. 7A-7C in panels (A), (B), and (C) depict a schematicrepresentation of an RPA reaction in progress.

FIGS. 8A-8C depict (A) examples of double stranded primers; (B) doublestranded primers after elongation and after annealing of the secondmember of a primer pair; (C) after the elongation of the second memberof a primer pair with the non-invading strand displaced.

FIG. 9 depicts investigation into the nature of double-stranded DNAtargets and targeting oligonucleotides. Experiments using eithersupercoiled templates or linearized DNAs suggest that recA catalyses theformation of intermediates capable of supporting polymerase elongationmost readily on supercoiled DNA or at the ends of linearized DNA.Tester3bio oligonucleotide (SEQ ID NO: 90) is shown.

FIG. 10 depicts backfire synthesis. Backfire synthesis occurs when arecA-coated targeting oligonucleotide possessing a 5′ overhang invades aduplex DNA end in the presence of a suitable polymerase and dNTPs. Thisnew duplex region is stable to subsequent branch migration and can beutilised as a platform for other activities. Forward fire, that is theelongation from the invading oligonucleotide, can also occur.

FIG. 11 depicts uses of backfire synthesis. Backfire synthesis can beuseful because it generates a branch migration resistant structure thatcan be used for application other than normal oligonucleotide priming.Some examples are shown here including introduction of a nicking enzymetarget site, introduction of an RNA polymerase promoter, and the lineargeneration of short dsDNA fragments through successiveinvasion/synthesis/cleavage events.

FIG. 12 depicts single stranded binding proteins facilitate recombinaseinvasion and primer extension. Both E. coli SSB and T4 gp32 with anN-terminal His tag (gp32(N)) stimulate recA-mediated invasion/elongationon a linear DNA template.

FIG. 13 depicts the requirement for a minimal oligonucleotide length oroverhang for invasion and elongation during end targeting of lineartemplates. When the invading oligonucleotide is targeted at ends of alinearized template a minimal oligonucleotide length, or an overhang isneeded for invasion/elongation to occur.

FIG. 14 depicts paranemic and pletonemic joints. Schematic descriptionof the formation of paranemic and plectonemic joints by recombinationevents involving DNA ends or embedded sequences.

FIG. 15 depicts the effect of crowding agents. Crowding agents can alterthe reaction behaviour. In the presence of polyethylene glycols, gp32(N)and recA recombinase, multiple invasion events can be stimulated onsingle templates without the requirement for 5′ overhang in thetargeting oligonucleotide.

FIG. 16 depicts end targeted amplification using leading strand RPA.Amplification of a target DNA using end-directed oligonucleotides,recA(C) protein, and the Klenow fragment of E. coli DNA polymerase I.

FIG. 17 depicts leading strand RPA and limits of Klenow processivity.Limited amplification of an approximately 300 base pair fragment whenonly 0.5 fmoles of starting template is utilised with the recA protein,gp32(N), and the Klenow fragment of E. coli. Strong accumulation ofshorter products suggests that the poor processivity of Klenow (10-50nucleotides) may underlie the template concentration dependence of thereaction.

FIG. 18 depicts spacing dependence of RPA primers. There is an optimalinteroligonucleotide length for RPA when using the Klenow fragment of E.coli DNA polymerase I. The template (SEQ ID NO: 1) and EcoRI overhang(SEQ ID NO: 91) sequences are shown.

FIG. 19 depicts RPA products that are largely double stranded. RPAreaction can generate double-stranded DNA products as evidenced byagarose gel electrophoresis and restriction enzyme cleavage.

FIG. 20 depicts activity of a recA C-terminal truncation mutant. MutantrecA protein with a deletion of the C-terminal acidic peptide(recA(C)delta) can promote strand exchange and extension in a lineartemplate run-on assay.

FIG. 21 depicts modified gp32 proteins. Shown is a schematicrepresentation of the bacteriophage T4 gp32 proteins used in this studyand the position of various modification and mutations. Figure discloses“6×His” as SEQ ID NO: 81.

FIG. 22 depicts activity of gp32 protein. Modified gp32 proteins show avariety of activities in linear invasion/run-on assays.

FIG. 23 depicts invasion and extension using uvsX. Modified uvsX proteinwith a C terminal His tag (uvsX(C)), or an additional deletion of theC-terminal acidic peptide, stimulates invasion/extension in a lineartemplate run-on assay.

FIG. 24 depicts RPA using uvsX(C). The modified recombinase uvsX(C) cansupport DNA amplification in the presence of gp32(N), the Klenowfragment of E. coli DNA polymerase I and polyethylene glycol (PEG).

FIG. 25 depicts wild-type versus modified gp32. The modified version ofgp32, gp32(C) is qualitatively different to wild-type untagged gp32.

FIG. 26 depicts titration of gp32 and effect of uvsY. Titration of gp32reveals a requirement for a minimal quantity of gp32, and a requirementfor uvsY(N) protein when untagged gp32 is employed.

FIG. 27 depicts factors affecting reaction rate and noise. Shown is aschematic representation of the factors affecting reaction behaviour,particularly reaction rate and noise. The predicted effects andinteractions of gp32, uvsX, UvsY and PEG are suggested, with theconclusion that an optimal balance between reaction rate and noise mustbe struck.

FIG. 28 depicts effects of PEG. PEG can reduce the average length oflinear invasion/run-on products in an uvsX-mediated linear run-onexperiment in the presence of gp32(C).

FIG. 29 depicts DNA end directed invasion. Shown is a schematicdepicting possible outcomes of end-directed invasion events.

FIG. 30 depicts RPA in a complex sample. The amplification of specificDNA targets from human genomic DNA.

FIG. 31 depicts RPA sensitivity. The sensitivity of amplification ofspecific DNA targets from human genomic DNA.

FIG. 32 depicts RPA sensitivity and template independent artifacts. Thesensitivity of amplification of specific DNA targets from human genomicDNA, and the existence of competing template-independent primerartifacts.

FIG. 33 depicts how primer artifacts may arise. Shown is a schematicrepresentation of the possible mechanism by which primer artifacts mayarise.

FIG. 34 depicts primer artifact suppression. Shown in schematic aremethods to suppress primer artifacts.

FIG. 35 depicts the use of hairpin oligonucleotides to stimulateself-priming of displaced strands. Shown is a schematic diagram of anamplification using self-complementary hairpin oligonucleotidesdeliberately to stimulate self-priming of displaced strands.

FIG. 36 depicts conditions that enable highly efficient noise-freeamplification from complex DNA sources. The sensitivity of amplificationof specific DNA targets from human genomic DNA under optimisedconditions that reduce or eliminate primer artifacts.

FIG. 37 depicts a schematic representation of RPA method as shown in(A), (B), and (C).

FIG. 38 depicts (A) STR markers from two individuals (1 and 2, fatherand son) amplified with primer pairs for seven independent markers usingRPA conditions C4; (B) Titration of reaction components to determineconcentrations that support in vitro amplification.

FIG. 39 depicts (A) Size limits of RPA reactions; (B) Elongationefficiencies from embedded or end sequences; (C) Sensitivity of RPAreactions; (D) Human DNA of the indicated copy number amplified withprimers ApoB4 and Apo300 generating a 300 bp fragment using conditionsC2; (E,F) Human DNA from single individuals amplified with primersD18S51 5′ and 3′. Conditions employed were C2 in (E), and C4 in (F).

FIG. 40 depicts specificity of RPA reactions for: (A) Primers BsA3 andBsB3, which amplify a 380 bp fragment using conditions C3. An asteriskindicates the position of the expected reaction product, and an arrowindicates the position of the genomic DNA; (B,C) OligonucleotidesApo600bio and Apo300rev which generate a 345 bp fragment usingconditions C4; (D) Mixtures of reaction components assembled in theabsence of the indicated components, PEG and buffer. Primers used areindicated. Target DNA was human male genomic DNA at 150 copies/μl; (E)Oligonucleotides targeting three independent loci in human genomic DNAincubated with overlapping primer pairs of 25, 28, or 32 bases asindicated.

FIG. 41 Primer noise at low target copy number. The consequences ofreducing the starting copy density of a target in a typical RPA reactionare shown.

FIG. 42 Selection of optimal primers by combining a selection ofcandidate forward and reverse primers and testing the outcome at verylow start copy densities. Figure discloses SEQ ID NO: 92.

FIG. 43 Theoretical consideration of how primer noise initiates. FIG. 43(A) discloses SEQ ID NO: 93; (B) discloses SEQ ID NO: 93; (C) disclosesSEQ ID NO: 94; (D) discloses SEQ ID NOS 93 and 95, respectively, inorder of appearance; (E) discloses SEQ ID NO: 95; (F) discloses SEQ IDNO: 96; (G) discloses SEQ ID NO: 97; (H) discloses SEQ ID NO: 98; (I)discloses SEQ ID NOS 98 and 93, respectively, in order of appearance;and (J) discloses SEQ ID NO: 98.

FIG. 44 Oligonucleotide improvement strategies—overview of three generalschemes.

FIG. 45 Consequences of reducing primer length—short primers give lessproduct, but can still retain activity at less than 30 residues.

FIG. 46 Locked nucleic acids can function in RPA and exhibit significantdifferences in product accumulation, noise accumulation, and polymeraseconcentration requirement.

FIG. 47 Adding homopolymeric stretches to the 5′ ends of primers (SEQ IDNOS 66, 99-100, 69 and 101-102, respectively, in order of appearance)may alter nucleoprotein activity.

FIG. 48 Betaine reduces levels of product and noise.

FIG. 49 Combination strategies in which nucleoprotein filaments withdifferential activities are combined.

FIG. 50 Detection formats incorporating a ‘third’ primer for productenrichment

FIG. 51 Bead capture I. Schematic of experimental strategy in which athird probe enriches bona fide products derived from the target.

FIG. 52 Bead capture II. Experimental results demonstrating that a lowactivity nucleoprotein filament immobilized on a solid support canparticipate as a third primer and separate target amplicons from primernoise.

FIG. 53 Trehalose stabilizes lyophilizates to permit all componentsexcept buffered sample to remain active for at least 10 days at roomtemperature.

FIG. 54 Minor groove binding dyes SYBR green and SYBR gold can beincluded in RPA reactions and will sense the accumulation of DNAreaction products.

FIG. 55 Using SYBR green dye for quantitative assessment of Bacillussubtilis genomic DNA copy number can be made over four order ofmagnitude.

FIG. 56 Using SYBR green dye quantitative assessment of Bacillussubtilis genomic DNA copy number can be made over at least five ordersof magnitude.

FIG. 57 Quantitative real-time RPA responds excellently to titration ofhuman genomic DNA.

FIG. 58 The presence of single-strand bubbles in imperfect probe:targethybrids leads to enhanced overall duplex disruption in a dynamicrecombination environment

FIG. 59 Hybridization of amplicons to an array of potential lengthpolymorphism probes in a dynamic recombination environment leads toenrichment of hybrids of perfect or near-perfect match as compared toclassical hybridization, or non-dynamic recombination environment.

FIG. 60 Single tube reverse transcription RPA (RT-RPA) shows highsensitivity without significant optimisation

FIG. 61 dUTP can be included in RPA reactions to wholly or partiallyreplace dTTP thus providing a mechanism to develop effective carry-overcontamination control.

FIG. 62 A strategy for controlling carry-over contamination in RPAreactions.

FIG. 63 Format of a stand-alone RPA assay to determine the presence of aspecific nucleic acid comprising disposable amplification pouch andlateral flow strip to detect successful amplification.

FIG. 64 Compatibility of RPA with crude lysates of human blood indicatethe possibility that sophisticated sample preparations may be obviatedfor many samples. FIG. 64A depicts that RPA is capable of amplificationfrom whole human blood. Expected amplicon is indicated by arrow. FIG.64B depicts that template yield is enhanced by treatment of whole bloodwith a simple lysis buffer. For each of the experimental samples weexpect the total possible number of genome copies to be ˜1750. This iscompared to ˜500 genome copies of purified DNA used as control.

FIG. 65 Utility of real-time RPA analysis in optimising reactionenvironment; salt and PEG concentrations.

FIG. 66 Utility of real-time RPA analysis in optimising reactionenvironment; Magnesium concentrations.

FIG. 67 Primer sequences (SEQ ID NOS 46-47, 33 and 32, respectively, inorder of appearance) affect amplification rate behaviour—preliminaryanalysis suggests that ‘fast’ primers may be associated with low Gcontent, and high C or C/T content.

FIG. 68 Utility of real-time RPA analysis in optimising reactionenvironment; heterologous 5′ sequences appended to oligonucleotidesinfluence reaction behaviour. Figure discloses SEQ ID NOS 66, 99-100, 69and 101-102, respectively, in order of appearance.

FIG. 69 Utility of real-time RPA analysis in optimising reactionenvironment; heterologous 5′ sequences appended to oligonucleotidesinfluence reaction behaviour—not all pyrimidine-rich 5′ sequences driveexcellent kinetic behaviour. Figure discloses SEQ ID NOS 66, 103-105, 69and 106-108, respectively, in order of appearance.

FIG. 70 Utility of real-time RPA analysis in optimising reactionenvironment; heterologous 5′ sequences appended to oligonucleotidesinfluence reaction behaviour—not all pyrimidine-rich 5′ sequences driveexcellent kinetic behaviour. Figure discloses SEQ ID NOS 66, 99,109-112, 69, 101 and 113-116, respectively, in order of appearance.

FIG. 71 So-called ‘third probes’ may be employed to monitor RPAreactions and increase specificity. Candidate enzymes for processingspecific duplexes containing the target and probe.

FIG. 72 Enzymes that recognise abnormal features such as helixdistortions and damaged bases/abasic sites may be employed to processprobe/target hybrids.

FIG. 73 Fluorescent probes demonstrate significantly differentproperties in the RPA environment compared to standard environments usedin other amplification reactions. Figure discloses SEQ ID NOS 117-119,respectively, in order of appearance.

FIG. 74 A probe (SEQ ID NO: 80) containing a tetrahydrofuranyl residuein is efficiently cut and extended in an RPA environment containingamplified target, the E. coli Nfo enzyme, and polymerase.

FIG. 75 A probe containing a tetrahydrofuranyl residue in efficiently,rapidly, and specifically cut and extended in an RPA environmentcontaining amplified target, the E. coli Nfo enzyme, and polymerase.

FIG. 76 Examples of probe (SEQ ID NOS 120, 120, 121 and 121,respectively, in order of appearance) design for real-time RPA studiesusing the E. coli Nfo enzyme.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides for Recombinase-Polymerase Amplification(RPA)—a method for the amplification of target nucleic acid polymers. Italso provides for a general in vitro environment in which highrecombinase activity is maintained in a highly dynamic recombinationenvironment, supported by ATP. One benefit of RPA is that it may beperformed without the need for thermal melting of double-strandedtemplates. Therefore, the need for expensive thermocyclers is alsoeliminated. The present invention describes two related strategies bywhich RPA can be configured to permit exponential amplification oftarget nucleic acid polymers.

Throughout this specification, various patents, published patentapplications and scientific references are cited to describe the stateand content of the art. Those disclosures, in their entireties, arehereby incorporated into the present specification by reference.

Leading strand recombinase-polymerase amplification (lsRPA)

In leading strand Recombinase-polymerase Amplification (lsRPA)single-stranded, or partially single-stranded, nucleic acid primers aretargeted to homologous double-stranded, or partially double-stranded,sequences using recombinase agents, which would form D-loop structures.The invading single-stranded primers, which are part of the D-loops, areused to initiate polymerase synthesis reactions. A single primer specieswill amplify a target nucleic acid sequence through multiple rounds ofdouble-stranded invasion followed by synthesis. If two opposing primersare used, amplification of a fragment—the target sequence—can beachieved. LsRPA is described briefly in FIGS. 1 and 2.

The target sequence to be amplified, in any of the methods of theinvention, is preferably a double stranded DNA. However, the methods ofthe invention are not limited to double stranded DNA because othernucleic acid molecules, such as a single stranded DNA or RNA can beturned into double stranded DNA by one of skill in the arts using knownmethods. Suitable double stranded target DNA may be a genomic DNA or acDNA. An RPA of the invention may amplify a target nucleic acid at least10 fold, preferably at least 100 fold, more preferably at least 1,000fold, even more preferably at least 10,000 fold, and most preferably atleast 1,000,000 fold.

The target sequence is amplified with the help of recombinase agents. Arecombinase agent is an enzyme that can coat single-stranded DNA (ssDNA)to form filaments, which can then scan double-stranded DNA (dsDNA) forregions of sequence homology. When homologous sequences are located, thenucleoprotein filament (comprising the recombinase agent) strand invadesthe dsDNA creating a short hybrid and a displaced strand bubble known asa D-loop. Suitable recombinase agents include the E. coli RecA protein,the T4 uvsX protein, or any homologous protein or protein complex fromany phyla. Eukaryotic RecA homologues are generally named Rad51 afterthe first member of this group to be identified. Other non-homologousrecombinase agents may be utilized in place of RecA, for example as RecTor RecO. Recombinase agents generally require the presence of ATP,ATPγS, or other nucleoside triphosphates and their analogs. It ispreferred that recombinase agents are used in a reaction environment inwhich regeneration of targeting sites can occur shortly following around of D-loop stimulated synthesis. Completed recombination eventsinvolving recombinase disassembly will avoid a stalling of amplificationor very inefficient linear amplification of ssDNA caused by oscillatingsingle-sided synthesis from one end to the other.

Naturally, any derivatives and functional analogs of the recombinaseagent above may also function itself as a recombinase agent and thesederivatives and analogs are also contemplated as embodiments of theinvention. For example, a small peptide from recA, which has been shownto retain some aspects of the recombination properties of recA, may beused. This peptide comprises residues 193 to 212 of E. coli recA and canmediate pairing of single stranded oligos (Oleg N. Voloshin, LijangWang, R. Daniel Camerini-Otero, Homologous DNA pairing Promoted by a20-amino Acid Peptide Derived from RecA. Science Vol. 272 10 May 1996).

Since the use of ATPγS results in the formation of stableRecombinase-agent/dsDNA complexes that are likely incompatible withefficient amplification, it is preferable to use ATP and auxiliaryenzymes to load and maintain the Recombinase-agent/ssDNA primercomplexes. Alternatively, the limitations of the use of ATPγS may beovercome by the use of additional reaction components capable ofstripping recA bound to ATPγS from exchange complexes. This role mightbe played by helicases such as the RuvA/RuvB complex.

The terms ‘nucleic acid polymer’ or ‘nucleic acids’ as used in thisdescription can be interpreted broadly and include DNA and RNA as wellas other hybridizing nucleic-acid-like molecules such as those withsubstituted backbones e.g. peptide nucleic acids (PNAs), morpholinobackboned nucleic acids, locked nucleic acid or other nucleic acids withmodified bases and sugars.

Structurally similar to RNA, LNA monomers are bicyclic compounds thatbear a methylene linker that connects the nucleotide sugar ring's2′-oxygen to its 4′-carbon. LNA polymers obey standard base-pairingrules, but their physical properties make them suitable for mismatchdiscrimination applications. LNA are available from Exiqon (Denmark) orProligo (USA, Colorado).

One embodiment of the invention is directed to a method of performingRPA. The method comprises two steps. In the first step, the followingreagents are combined in a reaction: (1) at least one recombinase; (2)at least one single stranded DNA binding protein; (3) at least one DNApolymerase; (4) dNTPs or a mixture of dNTPs and ddNTPs; (5) a crowdingagent; (6) a buffer; (7) a reducing agent; (8) ATP or ATP analog; (9) atleast one recombinase loading protein; (10) a first primer andoptionally a second primer; and (11) a target nucleic acid molecule. Inthe second step, the reagents are incubated until a desired degree ofamplification is achieved.

The recombinase may be uvsX, recA or a combination of both. Therecombinase may also comprise a C terminal deletion of acidic residuesto improve its activity. While any recombinase concentration disclosedin the specification may be used, preferred recombinase concentrationsmay be, for example, in the range of 0.2-12 μM, 0.2-1 μM, 1-4 μM, 4-6μM, and 6-12 μM.

The single stranded DNA binding protein may be the E. coli SSB or the T4gp32 or a derivative or a combination of these proteins. gp32 derivativemay include, at least, gp32(N), gp32(C), gp32(C)K3A, gp32(C)R4Q,gp32(C)R4T, gp32K3A, gp32R4Q, gp32R4T and a combination thereof (SeeFIG. 13). The DNA binding protein may be present at a concentration ofbetween 1 μM and 30 μM.

The DNA polymerase may be a eukaryotic polymerase. Examples ofeukaryotic polymerases include pol-α, pol-β, pol-δ, pol-ε andderivatives and combinations thereof. Examples of prokaryotic polymeraseinclude E. coli DNA polymerase I Klenow fragment, bacteriophage T4 gp43DNA polymerase, Bacillus stearothermophilus polymerase I large fragment,Phi-29 DNA polymerase, T7 DNA polymerase, Bacillus subtilis Pol I, E.coli DNA polymerase I, E. coli DNA polymerase II, E. coli DNA polymeraseIII, E. coli DNA polymerase IV, E. coli DNA polymerase V and derivativesand combinations thereof. In a preferred embodiment, the DNA polymeraseis at a concentration of between 10,000 units/ml to 10 units/ml, such asbetween 5000 units/ml to 500 units/ml. In another preferred embodiment,the DNA polymerase lacks 3′-5′ exonuclease activity. In yet anotherpreferred embodiment, the DNA polymerase contains strand displacingproperties.

Any of the proteins mentioned in the methods of the invention isunderstood to also include its derivative. These proteins includes atleast the following: recombinases, polymerase, recombinase loadingprotein, single stranded DNA binding protein, accessory agents,RecA/ssDNA nucleoprotein filaments stabilizing agent and the like. Thederivative of these proteins include, at least, a fusion proteincomprising a C terminus tag, N terminus tag, or C and N terminus tags.Non-limiting examples of suitable sequence tags include 6-histidine(6×-His; HHHHHH; SEQ ID NO: 81), c-myc epitope (EQKLISEEDL; SEQ ID NO:82), FLAG® octapeptide (DYKDDDDK; SEQ ID NO: 83), Protein C(EDQVDPRLIDGK; SEQ ID NO: 84), Tag-100 (EETARFQPGYRS; SEQ ID NO: 85), V5epitope (GKPIPNPLLGLDST; SEQ ID NO: 86), VSV-G (YTDIEMNRLGK; SEQ ID NO:87), Xpress (DLYDDDDK; SEQ ID NO: 88), and hemagglutinin (YPYDVPDYA; SEQID NO: 89). Non-limiting examples of suitable protein tags include3-galactosidase, thioredoxin, His-patch thioredoxin, IgG-binding domain,intein-chitin binding domain, T7 gene 10, glutathione-S-transferase(GST), green fluorescent protein (GFP), and maltose binding protein(MBP). It will be understood by those in the art that sequence tags andprotein tags can be used interchangeably, e.g., for purification and/oridentification purposes. Accordingly, as used herein, the terms “Histag” and “hexahistidine tag” encompass all suitable sequence tags andprotein tags as known in the art and indicated in this paragraph.

The dNTPs includes, for example, dATP, dGTP, dCTP, and dTTP. In leadingand lagging strand RPA, ATP, GTP, CTP, and UTP may also be included forsynthesis of RNA primers. In addition, ddNTPs (ddATP, ddTTP, ddGTP andddGTP) may be used to generate fragment ladders. The dNTP may be used ata concentration of between 1 μM to 200·μM of each NTP species. A mixtureof dNTP and ddNTP may be used with ddNTP concentrations at 1/100 to1/1000 of that of the dNTP (1 μM to 200 μM).

The crowding agents used in the RPA include polyethylene glycol (PEG),dextran and ficoll. The crowding agent may be at a concentration ofbetween 1% to 12% by volume or 1% to 12% by weight of the reaction.While all PEGs are useful, preferred PEGs include PEG1450, PEG3000,PEG8000, PEG10000, PEG compound molecular weight 15000- to 20,000 (alsoknown as Carbowax 20M), and a combination thereof.

The buffer solution in a an RPA may be a Tris-HCl buffer, a Tris-Acetatebuffer, or a combination thereof. The buffers may be present at aconcentration of between 10 to 100 mM. The buffered pH may be between6.5 to 9.0. The buffer may further contain Mg ions (e.g., in the form ofMg Acetate) at a concentration between 1 to 100 mM with a concentrationof between 5 to 15 mM being preferred. One preferred Mg concentration is10 mM (Mg concentration or Mg Acetate concentration).

Reducing agents to be used include DTT. The DTT concentration may bebetween 1 mM and 10 mM.

The ATP or ATP analog may be any of ATP, ATP-γ-S, ATP-β-S, ddATP or acombination thereof. A preferred ATP or ATP analog concentration isbetween 1 and 10 mM.

Recombinase loading protein may include, for example, T4uvsY, E. colirecO, E. coli recR and derivatives and combinations of these proteins.One preferred concentration of these proteins is between 0.2 and 8 μM.

The primers used may be made from DNA, RNA, PNA, LNA, morpholinobackbone nucleic acid, phosphorothiorate backbone nucleic acid and acombination thereof. Combinations thereof in this can refers to a singlenucleic acid molecules which may contain one or more of one baseconnected to one of more of another base. Preferred concentration ofthese molecules may be in the range of between 25 nM to 1000 nM. In onepreferred embodiment, the primers may contain a non-phosphate linkagebetween the two bases at its 3′ end and is resistant to 3′ to 5′nuclease activity. In another embodiment, the primers may contain alocked nucleic acid at its 3′ ultimate base or 3′ penultimate base. Forexample, in a nucleic acid of the sequence 5′-AGT-3′, the T is the 3′ultimate base and the G is the 3′ penultimate base. The primers may beat least 20 bases in length or at least 30 bases in length. In onepreferred embodiment, the primers are between 20 to 50 bases in length.In another preferred embodiment, the primers are between 20 to 40 basesin length such as between 30 to 40 bases in length.

The primers may contain additional 5′ sequence that is not complementaryto the target nucleic acid. These 5′ sequence may contain, for example arestriction endonuclease recognition site. The primers may be partlydouble stranded with a single stranded 3′ end.

In addition, any nucleic acid of any of the methods of the invention maybe labeled with a detectable label. A detectable label include, forexample, a fluorochrome, an enzyme, a fluorescence quencher, an enzymeinhibitor, a radioactive label and a combination thereof.

The target nucleic acid may be single stranded or double stranded. It isknown that single stranded nucleic acids would be converted to doublestranded nucleic acid in the methods of the invention. The targetnucleic acid may be supercoiled or linear. The sequence to be amplified(target nucleic acid) may be in between other sequences. The sequence tobe amplified may also be at one end of a linear nucleic acid. In oneembodiment, the target nucleic acid is linear and not connected tonon-target nucleic acids. In other words, where the target nucleic acidis linear, it can be in any of the following formats:

1. [non-target nucleic acid]-[target nucleic acid]-[non-target nucleicacid]

2. [non-target nucleic acid]-[target nucleic acid]

3. [target nucleic acid]-[non-target nucleic acid]

4. [target nucleic acid]

It should be noted that the arrangement above is intended to representboth single stranded nucleic acids and double stranded nucleic acids.“1” may be described as a target nucleic acid molecule which is linearwith two ends and wherein both ends are linked to a non-target nucleicacid molecule. “2” may be described as a target nucleic acid moleculewhich is linear with two ends and wherein one end is linked to anon-target nucleic acid molecule. “3” may be described as a targetnucleic acid molecule which is a linear nucleic acid molecule (with nonon-target nucleic acid).

In another embodiment, the target nucleic acid may be a single-strandednucleic acid which is converted to a double stranded nucleic acid by apolymerase or a double stranded nucleic acid denatured by the action ofheat or chemical treatment.

The target nucleic acid may be of any concentration such as less than10,000 copies, less than 1000 copies, less than 100 copies, less than 10copies or 1 copy in a reaction. A reaction volume may be 5 μl, 10 μl, 20μl, 30 μl, 50 μl, 75 μl, 100 μl, 300 μl, 1 ml, 3 ml, 10 ml, 30 ml, 50 mlor 100 ml.

The reaction may be incubated between 5 minutes and 16 hours, such asbetween 15 minutes and 3 hours or between 30 minutes and 2 hours. Theincubation may be performed until a desired degree of amplification isachieved. The desired degree of amplification may be 10 fold, 100 fold,1000 fold, 10,000 fold, 100,000 fold or 1000000 fold amplification.Incubation temperature may be between 20° C. and 50° C., between 20° C.and 40° C., such as between 20° C. and 30° C. One advantage of themethods of the invention is that the temperature is not critical andprecise control, while preferred, is not absolutely necessary. Forexample, in a field environment, it is sufficient to keep the RPA atroom temperature, or close to body temperature (35° C. to 38° C.) byplacing the sample in a body crevice. Furthermore, the RPA may beperformed without temperature induced melting of the template nucleicacid.

In another embodiment of the invention, the RPA further compriseaccessory agents. These accessory agents includes helicase,topoisomerase, resolvase and a combination thereof which possessunwinding, relaxing, and resolving activities respectively on DNA. Theaccessory agents may also include RuvA, RuvB, RuvC, RecG, PriA, PriB,PriC, DnaT, DnaB, DnaC, DnaG, DnaX clamp loader, polymerase corecomplex, DNA ligase and a sliding clamp and a combination thereof. Thesliding claim may be E. coli β-dimer sliding clamp, the eukaryotic PCNAsliding clamp, or the T4 sliding clamp gp45 and a combination thereof.The accessory agents may include, in addition, DNA Polymerase IIIholoenzyme complex consisting of β-Clamp, DnaX Clamp Loader, and thePolymerase Core Complex. These latter accessory agents would allow theperformance of leading and lagging RPA.

In another embodiment, the RPA may be performed in the presence of aRecA/ssDNA nucleoprotein filaments stabilizing agent. Examples of suchstabilizing include RecR, RecO, RecF and a combination thereof. Thesestabilizing agents may be present at a concentration of between 0.01 μMto 20 μM. Other examples of stabilizing agents include the T4 uvsYprotein which stabilizes uvsX/ssDNA nucleoprotein complexes.

Other components of RPA include a system for ATP regeneration (convertADP to ATP). Such system may be, for example, phosphocreatine andcreatine kinase.

The RPA reaction may also include a system to regenerate ADP from AMPand a to convert pyrophosphate to phosphate (pyrophosphate).

In one preferred embodiment, the RPA reaction as listed above areperformed with E. coli components completely by using recA, SSB, recO,recR and E. coli polymerase.

In another preferred embodiment, the RPA reaction is performed with T4components by using uvsX, gp32, uvxY, and T4 polymerase.

In one preferred embodiment, RPA may be performed by combining thefollowing reagents: (1) a uvsX recombinase at a concentration of between0.2 to 12 μM; (2) a gp32 single stranded DNA binding protein at aconcentration between 1 to 30 μM; (3) a Bacillus subtilis DNA polymeraseI large fragment (Bsu polymerase) at a concentration between 500 to 5000units per ml; (4) dNTPs or a mixture of dNTPs and ddNTPs at aconcentration of between 1-300 μM; (5) polyethylene glycol at aconcentration of between 1% to 12% by weight or by volume; (6)Tris-acetate buffer at a concentration of between 1 mM to 60 mM; (7) DTTat a concentration of between 1 mM-10 mM; (8) ATP at a concentration ofbetween 1 mM-10 mM; (9) uvsY at a concentration of between 0.2 μM-8 μM;(10) a first primer and optionally a second primer, wherein said primersare at a concentration of between 50 nM to 1 μM; and (11) a targetnucleic acid molecule of at least one copy. After the reaction isassembled, it is incubated until a desired degree of amplification isachieved. This is usually within 2 hours, preferably within 1 hour, suchas, for example, in 50 minutes.

One advantage of the invention is that the reagents for RPA, with thepossible exception of the crowding agent and buffer, may be freeze dried(i.e., lyophilzed) before use. Freezed dried reagent offer the advantageof not requiring refrigeration to maintain activity. For example, a tubeof RPA reagents may be stored at room temperature. This advantage isespecially useful in field conditions where access to refrigeration islimited.

In one embodiment, the RPA reagents may be freeze dried onto the bottomof a tube, or on a bead (or another type of solid support). To performan RPA reaction, the reagents are reconstituted in a buffer solution andwith a crowding reagent, or simply a buffered solution or water,dependant on the composition of the freeze-dried reagents. Then a targetnucleic acid, or a sample suspected to contain a target nucleic acid isadded. The reconstitution liquid may also contain the sample DNA. Thereconstituted reaction is incubated for a period of time and theamplified nucleic acid, if present, is detected.

Detection may be performed using any method, such as, for example, usingelectrophoresis on an agarose or PAGE gel followed by ethidium bromidestaining.

In any of the methods of the invention, the reagents that can be freezedried before use would include, at least, the recombinase, the singlestranded DNA binding protein, the DNA polymerase, the dNTPs or themixture of dNTPs and ddNTPs, the reducing agent, the ATP or ATP analog,the recombinase loading protein, and the first primer and optionally asecond primer or a combination of any of these.

In one preferred embodiment, the reagents are assembled by combining thereagents such that when constituted, they will have the followingconcentrations: (1) a uvsX recombinase at a concentration of between 0.2to 12 μM; (2) a gp32 single stranded DNA binding protein at aconcentration between 1 to 30 μM; (3) a T4 gp43 DNA polymerase or Bsupolymerase at a concentration between 500 to 5000 units per ml; (4)dNTPs or a mixture of dNTPs and ddNTPs at a concentration of between1-300 μM; (5) DTT at a concentration of between 1 mM-10 mM; (6) ATP at aconcentration of between 1 mM-10 mM; (7) uvsY at a concentration ofbetween 0.2 μM-8 μM. Optionally, a first primer and optionally a secondprime may be added where their concentration would be between 50 nM to 1μM when reconstituted. The reagents are freeze dried before use.Stabilizing agents such as trehalose sugar may be included in the freezedried mixture, for example at 20 mM to 200 mM and most optimally 40 mMto 80 mM in the reconstituted reaction, in order to improvefreeze-drying performance and shelf life. If desired, the freeze driedreagents may be stored for 1 day, 1 week, 1 month or 1 year or morebefore use.

In use, the reagents are reconstituted with buffer (a) Tris-acetatebuffer at a concentration of between 1 mM to 60 mM; and (b) polyethyleneglycol at a concentration of between 1% to 12% by weight or by volume,or (c) with water. If the primers were not added before freeze drying,they can be added at this stage. Finally, a target nucleic acid, or asample suspected of containing a target nucleic acid is added to beginthe reaction. The target, or sample, nucleic acid may be containedwithin the reconstitution buffer as a consequence of earlier extractionor processing steps. The reaction is incubated until a desired degree ofamplification is achieved.

Any of the RPA reaction conditions discussed anywhere in thisspecification may be freeze dried. For example, the following reagentscan be assembled by combining each reagent such that when constituted,they will have the following concentrations: (1) 100-200 ng/μl uvsXrecombinase; (2) 600 ng/μl gp32; (3) 20 ng/μl Bsu polymerase or T4polymerase; (4) 200 μM dNTPs; (5) 1 mM DTT (6) 3 mM ATP or an ATPanalog; (7) 16 ng/μl to 60 ng/μl uvsY; (8) 50 nM to 300 nM of a firstprimer and 50 nM to 300 nM of a second primer; (9) 80 mM Potassiumacetate; (10) 10 mM Magnesium acetate; (11) 20 mM Phosphocreatine; (12)50 ng/μl to 100 ng/μl Creatine kinase. The reagents may be freeze driedonto the bottom of a tube or in a well of a multi-well container. Thereagent may be dried or attached onto a mobile solid support such as abead or a strip, or a well.

In use, the tube with the reagent may be reconstituted with (1)Tris-acetate buffer at a concentration of between 1 mM to 60 mM andpolyethylene glycol at a concentration of between 1% to 12% by weight orby volume. If the reagents were dried or attached to a mobile solidsupport, the support may be dropped in a tube and reconstituted. Asdiscussed above, the primers may be dried as part of the reagent oradded after reconstitution. Finally, a target nucleic acid, or a samplesuspected of containing a target nucleic acid is added to begin thereaction. The reaction is incubated until a desired degree ofamplification is achieved.

As another example, the following reagents can be assembled by combiningeach reagent such that when constituted, they will have the followingconcentrations: (1) 100-200 ng/μl uvsX recombinase; (2) 300-1000 ng/μlgp32; (3) 10-50 ng/μl Bsu polymerase or T4 polymerase; (4) 50-500 μMdNTPs; (5) 0.1 to 10 mM DTT; (6) 3 mM ATP or an ATP analog; (7) 16 ng/μlto 60 ng/μl uvsY; (8) 50 nM to 1000 nM of a first primer and 50 nM to1000 nM of a second primer; (9) 40 mM to 160 mM Potassium acetate; (10)5 mM to 20 mM Magnesium acetate; (11) 10 mM to 40 mM Phosphocreatine;(12) 50 ng/μl to 200 ng/μl Creatine kinase. These reagents are freezedried and stored. In use, the reagents are reconstituted withTris-acetate buffer at a concentration of between 1 mM to 60 mM andpolyethylene glycol at a concentration of between 1% to 12% by weight orby volume. The primers, item 8 above, may be omitted before freezedrying and added after reconstitution. To initiate the RPA, a targetnucleic acid, or a sample suspected of containing a target nucleic acidis added. The reaction is incubated until a desired degree ofamplification is achieved.

Another embodiment of the invention comprises a kit for performing RPA.The kit may comprise any of the reagents discussed above for RPA in theconcentrations described above. The reagents of the kit may be freezedried. For example, the kit may contain (1) 100-200 ng/μl uvsXrecombinase; (2) 300 ng/μl to 1000 ng/μl gp32; (3) 10 ng/μl to 50 ng/μlBsu polymerase or T4 polymerase; (4) 50 μM to 500 μM dNTPs; (5) 0.1 to10 mM DTT; (6) 1 mM to 5 mM ATP or an ATP analog; (7) 16 ng/μl to 60ng/μl uvsY; (8) 50 nM to 1000 nM of a first primer and 50 nM to 1000 nMof a second primer (optional); (9) 40 mM to 160 mM Potassium acetate;(10) 5 mM to 20 mM Magnesium acetate; (11) 10 mM to 40 mMPhosphocreatine; (12) 50 ng/μl to 200 ng/μl Creatine kinase.

In a preferred embodiment, RPA is performed with several auxiliaryenzymes that can promote efficient disassembly ofRecombinase-agent/dsDNA complexes after DNA synthesis initiation. Theseauxiliary enzymes include those that are capable of stimulating 3′ to 5′disassembly and those capable of supporting 5′ to 3′ disassembly.

Auxiliary enzymes include several polymerases that can displace RecA inthe 3′ to 5′ direction and can stimulate 3′ to 5′ disassembly ofRecombinase-agent/dsDNA complexes (Pham et al., 2001). These DNApolymerase include E. coli PoIV and homologous polymerase of otherspecies. Normally in the life of E. coli, displacement of RecA in the 3′to 5′ direction occurs as part of SOS-lesion-targeted synthesis inconcert with SSB, sliding clamps and DNA polymerase. The polymeraseessential for this activity in E. coli is Poly, a member of the recentlydiscovered superfamily of polymerases including UmuC, DinB, Rad30, andRev1, whose function in vivo is to copy DNA lesion templates. Criticalto RPA, the in vitro 3′ to 5′ disassembly of RecA filaments cannot becatalyzed by PolI, PolIII, or PolIV alone. Only PolV, in concert withSSB, has measurable ATP-independent 3′ to 5′ RecA/dsDNA disassemblyactivity. In effect, PolV pushes and removes RecA from DNA in a 3′ to 5′direction ahead of the polymerase (Pham et al., 2001; Tang et al.,2000). Inclusion of PolV or a functional homologue may improve theamplification efficiency.

Other auxiliary enzymes include a class of enzymes called helicases thatcan be used to promote the disassembly of RecA from dsDNA. These promotedisassembly in both the 5′ to 3′ and 3′ to 5′ directions. Helicases areessential components of the recombination process in vivo and functionto move the branch points of recombination intermediates from one placeto another, to separate strands, and to disassemble and recyclecomponents bound to DNA. After the first round of invasion/synthesis hasoccurred in RPA, two new DNA duplexes are “marked” by the presence ofRecA bound over the site to which primers must bind for additionalrounds of synthesis. In such a situation dsDNA tends to occupy the highaffinity site in RecA, or homologs, until it is actively displaced,either by ATP hydrolysis-dependent dissociation in the 5′ to 3′direction, which may be limiting, or by 3′ to 5′ dissociation by someother active process. An ideal helicase complex for stimulatingdisassembly of RecA from intermediates consists of the E. coli proteinsRuvA and RuvB. The RuvAB complex promotes branch migration, anddissociates the RecA protein, allowing RecA to be recycled (Adams etal., 1994). Normally, the RuvAB complex is targeted to recombinationintermediates, particularly Holliday junction-like structures. As itworks the RuvAB complex encircles DNA and forces RecA from the DNA in anATP-driven translocation (Cromie and Leach, 2000; Eggleston and West,2000). This RecA dissociation activity has been demonstrated usingsupercoiled dsDNA bound with RecA, which does not even possess Hollidayjunctions (Adams et al., PNAS 1994). The RuvAB complex can recognizebranched structures within the RecA coated DNA. Incorporation of RuvABinto the RPA mixture will promote the dissociation of RecA from dsDNAfollowing strand exchange and displacement, allowing renewed synthesisof the duplicated template from the same site. Additionally, the RuvABcomplex can act in concert with RuvC, which finally cuts and resolvesHolliday junctions. With RuvC added to the RPA reaction mixture,complicated structures such as Holliday junctions formed at invasionsites, can be resolved. Resolvase activity, such as that provided byRuvC, is particularly important when the targeting oligonucleotides arepartially double-stranded. In such situations reverse branch migrationcan generate Holliday junctions, which can then be resolved by theRuvABC complex, to generate clean separated amplification products.

Still other auxiliary enzymes include the E. coli RecG protein. RecG canstimulate disassembly of branch structures. In vivo this proteinfunctions to reverse replication forks at sites of DNA damage byunwinding both leading and lagging strands driving the replication forkback to generate a 4-way junction (Cox et al., 2000; Dillingham andKowalczykowski, 2001; Singleton et al., 2001). In vivo such junctionsfunction as substrates for strand switching to allow lesion bypass. Invitro RecG will bind to D-loops, and will lead to a decrease in D-loopstructures by driving reverse branch migration. RecG prefers a junctionwith double-stranded elements on either side, hence partlydouble-stranded targeting oligonucleotides, homologous to the targetingsite in both single-stranded and double-stranded regions, would beideal. This would stimulate reverse branch migration and formation of aHolliday junction, which can be resolved by the RuvABC complex. In vivoRecG and RuvAB may compete to give different outcomes of recombinationsince branch migration will be driven in both directions (McGlynn andLloyd, 1999; McGlynn et al., 2000). In both cases the proteins targetjunction DNA coated with RecA, and disassemble it in an active manner.

Other auxiliary enzymes useful in an RPA reaction mixture are those thatallow continual generation of RecA nucleoprotein filaments in thepresence of ATP and SSB. In order to allow removal of RecA at theappropriate moments, it is preferred to use ATP rather than ATPγS in anRPA reaction. Unfortunately RecA/ssDNA filaments formed with ATPspontaneously depolymerize in the 5′ to 3′ direction, and in thepresence of SSB, as required here, repolymerization will not occur atsignificant rates. The solution to this problem is the use of the RecO,RecR, and possibly RecF proteins. Alternatively the uvsY protein may beemployed to stabilize the T4 uvsX nucleoprotein filaments in a similarmanner. In the presence of SSB and ATP, RecA/ssDNA filaments dissociate(Bork et al., 2001; Webb et al., 1995; Webb et al., 1997; Webb et al.,1999). If RecA/ssDNA is incubated in the presence of RecO and RecRproteins this dissociation does not occur. Indeed the RecR proteinremains associated with the filament and stabilizes the structureindefinitely. Even if ssDNA is bound by SSB, in the presence of RecR andRecO, filaments of RecA can reassemble displacing SSB. In the T4 phagesystem similar properties are attributed to the uvsY protein. Thus it ispossible to obviate the use of ATPγS, if necessary, by using ATP in thepresence of RecO and RecR to maintain RecA/ssDNA filament integrity, oruvsY to maintain the uvsX/ssDNA filament integrity. The RecF proteininteracts with the RecO and RecR system in a seemingly opposing manner.RecF competes with RecR tending to drive filament disassembly in vitro.It is likely that all three components in vivo function together tocontrol the generation of invading structures, while limiting the extentof RecA coating of ssDNA. In another preferred embodiment, RecF isincluded in RPA reactions at an appropriate concentration tore-capitulate the dynamics of the in vivo processes. In addition, RecFmay facilitate dissociation of RecA-coated intermediates after invasionhas occurred.

As described, the use of ATP rather than ATPγS, and/or the use ofdisplacing polymerases and helicases (e.g. the RuvA/RuvB complex), RecO,RecR and RecF, or alternatively the T4 uvsX recombinase with the uvsYprotein, should permit exponential amplification of double-stranded DNAby driving continual regeneration of targeting sites. This method,however, remains responsive to differences in initiation rate that mightoccur at the two opposing targeting sites. Such differences may lead toa decrease in amplification efficiency, and to the production of somesingle-stranded DNA. The PCR method largely avoids these complicationsbecause temperature cycling leads to coordinated synthesis from eitherside. In another embodiment, a situation analogous to the PCR conditionjust described may be induced by using temperature sensitive (ts)mutants of RecA that are non-functional at 42° C., but function at lowertemperatures in the range 25 to 37° C. (Alexseyev et al., 1996; Hicksonet al., 1981). In this case, synthesis from either end can besynchronized by periodic lowering to the permissive temperature and thenraising the reaction to a temperature non-permissive for the mutant RecAprotein function, but permissive for the other components. By performingRPA with tsRecA mutants in combination with cycling of reactiontemperatures, the number of molecules of DNA produced can be controlled.While this will require some mechanism to provide temperature cycling,the temperatures are well below those that would require the use ofthermophile-derived proteins. Indeed, a simple chemical-based orportable low-power temperature-cycling device may be sufficient tocontrol such reaction cycles.

RPA, as all other present-day nucleic acid amplification methods,employs polymerases to generate copies of template nucleic acidmolecules. It is a necessity of most nucleic acid polymerases thatincorporation requires a free 3′-hydroxyl moiety on the terminal sugarof a short stretch of double-stranded nucleic acid adjacent to the siteof new synthesis. This stretch of double-stranded nucleic acid istypically formed on a template by a short complementary sequence, calleda primer, which serves as an initiation site for the polymerasesynthesis reaction. In some cases a 3′ modification, such as asulfhydryl, may utilized to prime the synthesis reaction. The primernucleic acid, which is base-paired with the template and extended by thepolymerase, can be RNA or DNA. In vivo during genomic DNA replication,RNA primer sequences are synthesized de novo onto template DNA byprimase enzymes. Typically, for in vitro reactions the primer issupplied as a short, often chemically synthesized, single-stranded DNA(or modified DNA or RNA), and is usually referred to as anoligonucleotide primer. The primer is often of a specific sequence,although random primers can also be used. The primer is targeted tocomplementary sequences by virtue of its specific base-pairing capacity.Formation of hybrids between the oligonucleotide primer and targetnucleic acid are typically formed by incubation of the two in solutionunder conditions of salt, pH, and temperature that allow spontaneousannealing.

In the case of the PCR the oligonucleotide primer is usually in vastexcess for two main reasons. First, the high concentration will driverapid annealing. Second, as the reaction proceeds through rounds ofmelting, annealing and extension the primer is consumed and becomeslimiting. PCR targeted nucleic acids are often initially double-strandedin character, and if not, become double stranded following the firstsynthetic cycle. Such double-stranded molecules cannot anneal newoligonucleotides at temperature and solvent conditions appropriate forthe catalytic activity and stability of most prokaryotic and eukaryoticproteins. Consequently, in order to allow cycles of amplification theoriginal template and the newly synthesized strands must be firstseparated before annealing can occur once again. In practice this isachieved by thermal melting. For PCR, temperatures of at least 80° C.are required for thermal melting of most double-stranded nucleic acidmolecules of lengths greater than 100 base pairs. In most PCR protocolsa temperature of 90 to 100° C. is applied to melt the DNA. Suchtemperatures allow only rare thermostable enzymes to be used. Thesepolymerases are typically derived from thermophilic prokaryotes.

The advantage of RPA is that it allows the formation of short stretchesof double-stranded nucleic acids bearing a free 3′--OH for extensionfrom double-stranded templates without thermal melting. This is achievedby using the RecA protein from E. coli (or a RecA relative from otherphyla including the T4 uvsX protein). In the presence of ATP, dATP,ddATP, UTP, ATPγS, and possibly other types of nucleoside triphosphatesand their analogs, RecA or uvsX will form a nucleoprotein filamentaround single-stranded DNA. This filament will then scan double-strandedDNA. When homologous sequences are located the recombinase will catalyzea strand invasion reaction and pairing of the oligonucleotide with thehomologous strand of the target DNA. The original pairing strand isdisplaced by strand invasion leaving a bubble of single stranded DNA inthe region.

RecA protein can be obtained from commercial sources. Alternatively itcan be purified according to standard protocols e.g. (Cox et al., 1981;Kuramitsu et al., 1981). RecA homologues have been purified fromthermophilic organisms including Thermococcus kodakaraensis (Rashid etal., 2001), Thermotoga maritima (Wetmur et al., 1994), Aquifexpyrophilus (Wetmur et al., 1994), Pyrococcus furiosus (Komori et al.,2000), Thermus aquaticus (Wetmur et al., 1994), Pyrobaculum islandicum(Spies et al., 2000), and Thermus thermophilus (Kato and Kuramitsu,1993). RecA has also been purified from other prokaryotes e.g.Salmonella typhimurium (Pierre and Paoletti, 1983), Bacillus subtilis(Lovett and Roberts, 1985), Streptococcus pneumoniae (Steffen andBryant, 2000), Bacteroides fragilis (Goodman et al., 1987), Proteusmirabilis (West et al., 1983), Rhizobium meliloti (Better and Helinski,1983), Pseudomonas aeruginosa (Kurumizaka et al., 1994), from eukaryotese.g. Saccharomyces cerevisiae (Heyer and Kolodner, 1989), Ustilagomaydis (Bennett and Holloman, 2001), including vertebrates e.g. HumanRad51 (Baumann et al., 1997) and Xenopus laevis (Maeshima et al., 1996),as well as plants including broccoli (Tissier et al., 1995). We herealso show that E. coli recA, and T4 uvsX protein, can be purified fromoverexpression cultures using a hexahistidine tag at the C terminus, andremain biologically active. This is of great utility for the productionof recombinant protein.

For clarity of description, leading strand Recombinase-PolymeraseAmplification method (lsRPA) can be divided into four phases.

1) Sequence Targeting RPA is initiated by targeting sequences usingsynthetic oligonucleotides coated with RecA, or a functional homologuesuch as the T4 uvsX protein. In order to permit exponentialamplification two such synthetic oligonucleotides would be employed in amanner such that their free 3′-ends are orientated toward one another.Nucleoprotein filaments comprising these oligonucleotides andrecombinase protein will identify targets in complex DNA rapidly andspecifically. Once targeted the recombinase protein catalyses strandexchange such that D-loop structures are formed. It may be necessary touse ATP rather than ATPγS in the procedure for efficient amplification.If ATP is used, RecO, RecR, and/or RecF, molecules may prove essentialfor efficient amplification, or uvsY protein if uvsX recombinase isemployed.

2) Initiation of DNA Synthesis

DNA polymerases will detect and bind to the hybrid between the invadingoligonucleotides and the template DNA and initiate DNA synthesis fromthe free 3′-hydroxyl exposed in the hybrid. Exposure of this3′-hydroxyl, and subsequent DNA synthesis, will likely requiredisassembly of recombinase protein from the double-stranded hybridformed by strand exchange. To attain this disassembly it will probablybe necessary to employ ATP, which can support spontaneous disassembly ofrecombinase from invasion complexes. Additionally disassembly can bestimulated/enhanced by the use of other proteins contained within thereaction mixture such as RuvA, RuvB, RuvC, recG, other helicases, orother stimulatory components, which can act to strip recombinase fromthe strand exchange product.

3) Strand Displacement DNA Synthesis and Replicon Separation.

As the DNA polymerases synthesize complementary copies of template DNAsusing the free 3′-hydroxyls of invading oligonucleotides, or theirpartly extended products, the polymerases displace single-stranded DNAs,which may be coated with single strand binding proteins (SSB) includedin the reaction. In an ideal configuration, invasion of oligonucleotidesat both ends of the target nucleic acid sequence will occur in similartimeframes, such that two polymerases on the same template nucleic acidwill initially progress toward one another. When these extendingcomplexes meet one another, the original template should simply fallapart, and the polymerases will continue to synthesize without a needfor strand displacement, now copying SSB-bound ssDNA template. Becauseof steric hinderance, polymerases may become dissociated from thetemplate temporarily when the polymerases meet to permit the separationof the two template strands

4) Completion of Synthesis and Re-Invasion.

Once the template strands have separated, polymerases can complete theextension to the end of the template (or past the sequence acting as thesecond, facing, targeting site if the initial template is longer thanthe desired product). To permit exponential amplification it isnecessary for new products to be targeted and replicated in a mannersimilar to the original templates, that is from both targeted ends. Thenewly synthesized targeted site will be freely available to targetingrecombinase/oligonucleotide filaments. The site initially used to primesynthesis should also have been freed as a consequence of the use ofconditions in the reaction that favor disassembly of recombinase fromstrand exchange products. Providing the re-invasion at this latter siteoccurs in less time than it takes the polymerase to synthesize past thesecond targeting site, be primed at that second site, and return to thefirst site, then single-stranded DNA will not be the primary product andexponential amplification will occur. Having multiple syntheticcomplexes operating on the same template raises the possibility thatvery short amplification times can be achieved.

Recombinase-Polymerase Amplification (RPA) Using Simultaneous Leadingand Lagging Strand Synthesis

In our description of (leading strand RPA) IsRPA we detail amulti-component system with the capacity to regenerate targetingsequences thus permitting exponential amplification of double-strandedDNA. Unlike the Zarling method, lsRPA avoids the linear production ofsingle-stranded DNA. There is another approach to solving this problemthat completely avoids the possibility of single-stranded products and arequirement for simultaneous end initiation. This method necessarilyinvolves a more complex reaction mixture. Nevertheless all of therequired components are now well understood and should be amenable toassembly into a single system. This system will recapitulate eventsoccurring during the normal replication cycle of cells to permit coupledleading and lagging strand synthesis. This method, leading/laggingstrand RPA is described briefly in FIGS. 1 and 3.

During normal replication in vivo, double-stranded DNA is simultaneouslyseparated into 2 strands and both are copied to give 2 new molecules ofdouble-stranded DNA by a replication machine. This ‘machine’ couplesconventional 5′ to 3′ leading strand synthesis with lagging strandsynthesis, in which short RNA primers are synthesized onto templatenucleic acids by primase enzymes. During lagging strand synthesis, shortfragments of DNA are produced, called Okazaki fragments, which areligated together to form contiguous lagging strands. This simultaneousleading-strand/lagging-strand synthesis is responsible for duplicationof the entire genome of prokaryotic and eukaryotic organisms alike. Theessential components of this system have been identified andcharacterized biochemically. The components can be assembled in vitro toachieve a more efficient amplification than possible using onlyleading-strand synthesis.

The essential components of the replication ‘machine’ are now wellcharacterized for E. coli and certain other organisms such as T4 phage.This machine comprises the PolIII holoenzyme (Glover and McHenry, 2001;Kelman and O'Donnell, 1995) and the primosome (Benkovic et al., 2001;Marians, 1999). The PolIII holoenzyme is made up of ten polypeptidecomponents. Each holoenzyme contains two, asymmetrically oriented, corestructures, each consisting of a polymerase (a subunit) and twoadditional core components the a subunit, which possesses 3′ to 5′exonuclease activity, and the 0 subunit. In addition to the core complexanother set of polypeptides provide the holoenzyme with processivity andcouple leading and lagging strand synthesis. The β-dimer sliding clampencircles the template DNA affixing the complex to the template withextremely high affinity. The sliding clamp loaded onto DNA by the DnaXclamp loader comprising the τ2γδδδχψpolypeptide subunits.

For clarity of description, the RPA method can be divided into fourphases. In reality all phases will occur simultaneously in a singlereaction.

1) Sequence Targeting

RPA is initiated by targeting sequences using synthetic oligonucleotidescoated with RecA, or T4 uvsX, or a functional homologue. Suchnucleoprotein filaments will identify targets in complex DNA rapidly andspecifically. Once targeted, the RecA or uvsX protein catalyses strandexchange such that a D-loop structure is formed. It may be necessary touse ATP rather than ATPγS in the procedure for efficient amplification.The linkage of leading and lagging strand syntheses however may obviatethe requirement for very rapid recombinase stripping after initiation ofsynthesis. If ATP is used, RecO, RecR, and RecF may need to be employedwith bacterial recA recombinase, or the T4 uvsY, proteins may proveessential for efficient amplification with T4 uvsX protein.

2) Primosome Assembly

Primosomes can be assembled at D-loops. Normally, in E. coli, D-loopstructures are formed by RecA as part of the mechanism to rescue damagedDNA in vivo, or during other forms of recombination. The purpose of thecombined action of RecA-mediated strand exchange and primosome assemblyis to generate a replication fork. A replication fork is thenucleoprotein structure comprising the separated template DNA strandsand the replisome. The replisome consists of the polymerase holoenzymecomplex, the primosome, and other components needed to simultaneouslyreplicate both strands of template DNA. Primosomes provide both the DNAunwinding and the Okazaki fragment priming functions required forreplication fork progression. Similar primosome assembly occurs atrecombination intermediates in T4 phage directed by gp59 and gp41protein.

Primosome assembly has been studied intensively through genetic andbiochemical analysis in E. coli. The minimal set of polypeptidesrequired for this process is well known and exist as purifiedcomponents. The primosome assembly proteins are PriA, PriB, PriC, DnaT,DnaC, DnaB, and DnaG. These proteins have been shown sufficient toassemble a primosome complex on bacteriophage .PHI.X174 DNA in vitro(Kornberg and Baker, 1992; Marians, 1992). PriA binds to the primosomeassembly site (PAS) on the .PHI.X174 chromosome. Then PriB, DnaT, andPriC bind sequentially to the PriA-DNA complex. PriB appears tostabilize PriA at the PAS and facilitate the binding of DnaT (Liu etal., 1996). PriC is only partially required for the full assemblyreaction. Omission of PriC from the reaction will lower priming 3 to 4fold (Ng and Marians, 1996a; Ng and Marians, 1996b). The function ofPriC in the bacterium is genetically redundant to PriB. DnaC then loadsDnaB into the complex in an ATP-dependent fashion. This PriABC-DnaBTcomplex is competent to translocate along the chromosome. The DnaGprimase can interact transiently with the complex to synthesize RNAprimers.

During replication in E. coli, DnaB and DnaG function as a helicase andprimase respectively. These two components are continually required inassociation with the PolIII holoenzyme to synthesize primers for theOkazaki fragments. Hence, DnaB and DnaG are the core components of themobile primosome associated with the replication fork. The otherprimosome components described are essential for assembly of theprimosome onto DNA, and for associating a dimeric polymerase. Theprimosome assembly proteins are required for the re-establishment ofreplication forks at recombination intermediates formed by RecA andstrand exchange. PriA can initiate assembly of a replisome, competentfor DNA synthesis, on recombination intermediates. It is possible totarget D-loops in vitro with a mixture of PriA, PriB, and DnaT, whichare then competent to incorporate DnaB and DnaC. Once a primosome hasbeen formed at the D-loop, all that remains to initiate replication isto load a holoenzyme complex to the site. Alternatively in the phage T4system the gp59 helicase loader protein recruits and assembles the gp41replicative helicase to D-loop structures

-   -   3) Fork Assembly and Initiation of DNA Synthesis

Replication forks will assemble at the site of primosome assembly. In E.coli the presence of a free 3′-end on the invading strand of the D-loopstimulates the DnaX clamp loader complex detailed earlier to assemble aβ-dimer at this site to act as a sliding clamp. The holoenzyme and 2core units are joined together by the scaffold τ subunit. The τ subunitalso has interaction surfaces for the β-dimer, for the clamp loader, andfor the DnaB helicase component of the primosome. These multipleinteractions are necessary to coordinate synthesis of both leading andlagging strands using the 2 asymmetrically joined core polymerasecomplexes. In T4 phage the gp59/41 proteins with uvsY and gp32 proteins,and with other components coordinate assembly of the sliding clamp gp45aided by gp44 and gp62 proteins initiates replisome assembly.

In E. coli the primosomal primase, DnaG, synthesizes a short RNA primeronto the unwound lagging strand DNA template. In the presence of theholoenzyme, the clamp loader recognizes the RNA/DNA duplex and loads asecond β-dimer clamp onto this site. The presence of an active primosomeand the interaction of the τ subunit with DnaB are critical to ensuresimultaneous leading/lagging strand synthesis. Without this interactionthe polymerase will move away from the primosome site without coupling.

A replication fork is now assembled. Synthesis of both leading andlagging strand will now occur simultaneously, and the DnaB helicase willseparate template strands ahead of the oncoming holoenzyme. The laggingstrand holoenzyme core will generate Okazaki fragments of 1 to 2kilobases in length. Once the lagging strand polymerase encounters theprevious RNA primer, it dissociates from the β-clamp and synthesis isinitiated from a newly assembled clamp loaded in the vicinity of thefront of the leading strand. The same lagging strand holoenzyme corewill be re-used since it is physically tethered to leading strand core.

There is a dynamic interaction between β-dimer clamps, core subunits,and clamp loaders. Their affinities can switch depending upon thephysical circumstances. The β-dimer that has been ‘abandoned’ at the endof the Okazaki fragments may be recycled via active removal by clamploaders, or excess δ subunit that may be present.

The RNA primers at the ends of Okazaki fragments are removed by the 5′to 3′ exonuclease activity of DNA polymerase I. DNA ligase then joinsthe Okazaki fragments together forming a continuous lagging strand.

4) Fork Meeting and Termination

In RPA, replication is initiated at two distant sites and thereplication forks are oriented toward each other. As replication forksconverge the two original template strands will dissociate from oneanother as they become separated entirely both behind, and in front, ofeach fork. The leading strand core of each fork will then completesynthesis, the remaining RNA primers will be processed, and the finalproducts will be two double-stranded molecules. We can reasonably expectto amplify DNA's on the order of several Megabases (Mb) by such anapproach. In this disclosure, megabase also encompasses megabasepairs.Based on the known synthetic rate of the PolIII holoenzyme we can expectthe replication forks to proceed at a rate of approximately 1 Mb/1000seconds, i.e., approximately 15 to 20 minutes per cycle for a 1 Mbfragment.

The final consideration is the mechanism by which rapid exponentialamplification of DNA will be achieved. The key to this process will beto allow efficient reinvasion of the targeting sites by the use ofmixtures of helicases, resolvases and the RecO, RecR, and RecF proteins.Under appropriate conditions reinvasion and primosome assembly should bepossible shortly after a holoenzyme has moved away from thefork-assembly site. Continual invasions should present no problems sincethe DNA will simply become branched at many points. Each branch willnaturally resolve as it encounters the oncoming fork. Under theseconditions it may be possible to achieve enormous amplification in timessimilar to the time taken to replicate the DNA only once. It may becritical however to limit the concentrations of targetingoligonucleotides to avoid nucleotide depletion prior to the completionof synthesis.

In addition to the holoenzyme complex, the replication machine employsanother complex known as the primosome, which synthesizes the laggingstrand and moves the replication fork forwards. The primosome complexcomprises a helicase encoded by DnaB and a primase encoded by DnaG.Finally, in addition to the proteins of the holoenzyme and primosome,replication requires the activity of single-stranded DNA binding protein(SSB), E. coli DNA polymerase I and DNA ligase. These latter twocomponents are required to process Okazaki fragments.

Nested RPA

In another embodiment, RPA amplification may be performed in a processreferred to herein as “nested RPA.” A difficulty in detecting a raresequence is that there can be a high ratio of non-target to targetsequence. The ability of a RPA to discriminate between target andnon-target DNA and amplify only target sequences is a key aspect ofimproved sensitivity. Discrimination between non-target and target is areflection of the specificity of the primers and reaction conditions.The more specific a reaction is the greater the relative amount of thespecific target sequence that is produced and the easier that product isto detect. An increase in specificity can, therefore, increasesensitivity as well.

The need for improved sensitivity and specificity can be addressed byusing nested RPA. The nested RPA involves a first RPA of a first regionof DNA. Then the reaction mixture is diluted, for example, by 10, 20,30, 40, 50, 75, or 100 fold or more to reduce the concentration of thefirst primer pair, and a second primer pair is introduced into thereaction mixture and RPA repeated. According to one embodiment of theinvention, the second primer pair is designed to be internal to thefirst primer pair to amplify a subsequence of the first RPA product. Themethod increases specific amplification, i.e., reduces non-specificbackground amplification products and therefore increases sensitivity.Such non-specific amplification products, although they arise by virtueof fortuitous partial homology to the flanking primers, are unlikely toalso have sufficient homology to the nested primers to continue toamplify. Detection and specificity of RPA may be further improved bylabeling one or both of the second primer pair such that only primersamplified with one or both of the second primer pair is detected.

Nested RPA is not limited to the use of two sets of primer. Naturally,more sets of primers may be used to increase specificity or sensitivity.Thus, three, four, or five pairs of primers may be used. Furthermore,the different sets of primers, as another embodiment of the invention,may share common primers as illustrated in FIG. 4.

In FIG. 4, the primer sets are designed to be used sequentially. Forexample, a first RPA is performed with primer set 1, a second RPA usingthe amplified product of the first RPA is performed with a primer set 2,a third RPA using the amplified product of the second RPA is performedwith a primer set 3, a fourth RPA using the amplified sequence of thethird RPA is performed with a primer set 4, and finally, a fifth RPA isperformed using the amplified product of the fourth RPA is performedwith a primer set 5. In this case, primer set 1, 2, and 3, share acommon primer-primer (a). Primer 3, 4, and 5 share a commonprimer-primer (b).

Nested RPA may be performed using any of the two RPA methods describedas well as a combination of the two methods in any particular order.That is, RPA may be performed solely by leading strand RPA, solely byleading and lagging strand RPA, or a combination of leading strand RPAand leading and lagging strand RPA in any particular order.

One benefit of any of the RPA methods of the invention is the size ofthe amplified product. While current methods of amplification such asPCR are limited to an upper limit of about 10 Kb, RPA methods arecapable of amplifying regions of nucleic acids of up to hundreds ofmegabases. For leading/lagging strand RPA, the sizes of a targetsequence to be amplified can be hundreds of megabases, such as, forexample, less than 500 megabases, less than 300 megabase, less than 100megabase, less than 70 megabase, less than 50 megabase, less than 25megabase, less than 10 megabase, less than 5 megabase, less than 2megabases, less than one megabase, less than 500 kb, less than 200 kb,less than 100 kb, less than 50 kb, less than 25 kb, or less than 10 kb,less than 5 kb, less than 2 kb, less than 1 kb. For lsRPA, the sizes ofa target sequence can be in the megabase range such as, less than 5megabase, less than 2 megabases, less than one megabase, less than 500kb, less than 200 kb, less than 100 kb, less than 50 kb, less than 25kb, or less than 10 kb, less than 5 kb, less than 2 kb, less than 1 kb.

General Considerations for Reconstituting and EnablingRecombinase-Mediated Amplification Reactions

Both lsRPA and leading/lagging RPA rely on similar use of recombinaseproteins to target oligonucleotide primers, however they differ in themode by which the new daughter duplexes are formed during amplification.In leading/lagging RPA a full replication fork is established thatsimultaneously synthesizes leading and lagging strands so that two newduplexes are concomitantly formed. In leading strand RPA (lsRPA), onlyleading strand synthesis occurs so that synthesis generates one duplexand one displaced single-stranded DNA as products.

In RPA DNA synthesis initiated after strand exchange is accomplished bya polymerase. During extension of the newly synthesised strand thepolymerase must be able to displace the outgoing strand, either alone orin combination with a helicase capable of mediating outgoing stranddisplacement. Extension of the invading primer results eventually in therelease of the outgoing strand as a single-stranded DNA. To ensure thatgeometric amplification occurs, and that the reaction produces a vastmajority of double-stranded DNA, it is necessary for this displacedsingle-stranded DNA to serve as template for DNA synthesis from theopposite direction. This is a central consideration for amplificationusing lsRPA. Two other central considerations are the polymerase speciesused, and the existence of a stable, dynamic, recombinase system thatfunctions efficiently in the presence saturating levels of single-strandbinding proteins. These considerations are important for both theleading/lagging RPA and lsRPA.

A) Ensuring Generation of Double-Stranded DNA from the Displaced Strand

The generation of the second strand of DNA in lsRPA can be achieved inone of several ways:

1) The displaced single-stranded DNA can simply hybridise to thecomplementary strand which has been displaced from invasion andextension of a second ‘facing’ targeting oligonucleotide. Alternativelythe displaced single-stranded DNA can hybridise directly with the second‘facing’ oligonucleotide. Such hybridisation events may occurspontaneously, or may be mediated by the strand assimilating activitiesof DNA binding proteins such as recombinases or single-stranded DNAbinding proteins. Following hybridisation a polymerase will extend fromthe free 3′ end to generate a double-stranded product. Note that forthis to occur efficiently the reaction environment must enablehybridisation of complementary single-stranded DNAs, a situation notalways compatible with the other aspects of the RPA reaction. In somecircumstances a hybridising oligonucleotide with a modified backbone,unable to interact with most DNA binding protein, could be used.

2) If strand-displacement synthesis begins simultaneously from opposingoligonucleotide primer on the same template then the two convergingreplication complexes will eventually meet somewhere in the middle ofthe template. Provided that these converging complexes are able to passone another the template strands will separate and each complex willcomplete replication by copying a single-stranded rather thandouble-stranded template with no further need for strand displacement.

3) If the outgoing strand possesses the capacity to form a hairpin thenself-priming second strand synthesis may occur. This activity wouldresult in a covalently linked duplex with a hairpin at one end, whichcould become a target for further invasion/extension reactions. Thissituation is not ideal for many applications, as it will generateproducts with variable lengths and structures. This may, however, beacceptable for detection assays, such as some diagnostic tests.Furthermore it may be possible to engineer primers such that after thefirst few rounds of invasion/extension most outgoing strands are capableof self-priming. This mode of duplex DNA formation may be veryefficient.

Which of these three general processes dominate in an lsRPA reactionwill depend on many factors. The most important factors are the distanceseparating the two oligonucleotides primers in the target, the invasionrate, and the sequence of the oligonucleotides and template.

In the second general format of RPA, leading/lagging RPA, the generationof substantial single-stranded DNA is avoided by establishing a fullreplication fork at the invasion site. A full replication fork permitsthe simultaneous copying of both leading and lagging strands (whichwould be equivalent to the outgoing strand). Leading/lagging RPA iselegant in its avoidance of the generation of single-stranded DNA,however a larger number of distinct proteins is required to generatefull replication forks. Nevertheless most aspects of optimisation forRPA reactions apply to both lsRPA and leading/lagging RPA.

B) Choice of Polymerase, or Polymerase/Helicase System

The lsRPA method is similar in some respects to PCR. Both processes useof pairs of oligonucleotide primers orientated with 3′ ends pointedtoward one another in the target DNA and both methods achieve geometricamplification through the use of reaction products as targets forsubsequent rounds of DNA synthesis. There are, however, fundamentaldifferences in the reaction configurations. For example, in RPA targetDNA is double-stranded prior to synthesis, whereas in PCR it issingle-stranded after thermal separation of strands. In RPA, DNAsynthesis must necessarily use DNA polymerases or polymerase complexescapable of strand displacement. Furthermore, because partially copiedstrands are physically associated with displaced strand through thetemplate, there is a risk that if the polymerase dissociates temporarilyfrom the template the 3′ end of the new strand, and eventually the wholenew strand, will be lost by the action of branch migration or anotherphenomenon known as bubble migration. This suggests that ideallyprocessive polymerases will be used for RPA reactions. It is alsoimportant to consider that if converging replication complexes cannotreadily pass one another without polymerase dissociation then someprocessive polymerases may inhibit the RPA reaction on some templates.In summary the ideal choice of polymerase will depend on the preciseformat and objective of the particular RPA reaction, in particular thesize of the product to be amplified.

C) Establishment of a Stable Persistent Active Recombinase Activity in aNoise-Suppressed Environment

A third consideration is how to establish a stable, but dynamic,recombinase activity, while silencing the noise generated by aberrantprimer annealing seen at low temperatures. This means establishing areaction environment that balances several seemingly incompatiblerequirements. For efficient RPA recombinase proteins must remain activewhile assembled into oligonucleotide/recombinase filaments scanning fortarget double-stranded DNAs. These complexes must also disassemble aftercompleting strand exchanges to permit DNA synthesis. This must happen inan environment rich in single-stranded DNA proteins. These proteins areneeded to stimulate recombination and DNA synthesis while preventingaberrant oligonucleotide behaviour by melting secondary structures.Fundamentally, the recombinases and single-stranded binding proteins arein competition for oligonucleotide binding. While the single-strandbinding proteins are necessary to enable efficient strand displacementduring synthesis, they suppress recombination activity because they havea higher affinity for single-stranded DNA and bind with morecooperativity than do recombinases.

Establishment of a functional recombinase/replication reactionenvironment requires nucleotide cofactors. RecA, and other recombinases,require nucleotide co-factors, such as ATP, to assemble filaments ontosingle-stranded DNA, perform homology searches, and complete strandexchange. We have surmised that non-hydrolysable analogues such asATP-γ-S would be incompatible with RPA because the extremely highstability of the 3-stranded DNA/recA intermediate formed in the presenceof with ATP-γ-S would prevent reinvasion at primer targets and wouldthus prevent efficient amplification. They may even prevent any usefulaccess of polymerase to the recombination intermediate. Earlier attemptsto amplify DNA using E. coli recA (Zarling et al) were probably limitedby the ATP-γ-S in the described reactions.

The requirement for ATP in the reaction and the fact thatrecombinase-complexes will be dynamically forming and disassemblingintroduces additional complexities, primarily due to complexinteractions and competition between key reaction components. Inparticular single-stranded binding proteins, such as the E. colisingle-stranded binding proteins, such as E. coli SSB or T4 phage gp32protein, are necessary to stimulate recombination by recA andhomologues, due both to their capacity to collect the outgoing strand,and to melt secondary structures in single-stranded DNAs thus enhancingrecombinase loading. In RPA it is likely that single-stranded bindingproteins will further stimulate DNA synthesis by binding and stabilisingthe displaced DNA strand, preventing undesirable branch migration.

Despite the clear requirement for single-stranded binding proteins,these proteins generally have a considerably higher affinity forsingle-stranded DNA than recombinases such as recA, or uvsX, and caninhibit nucleation of recombinase/DNA filaments. Moreover, as filamentsformed in the presence of ATP undergo end-dependant disassembly (Bork,Cox and Inman J Biol Chem. 2001 Dec. 7; 276(49):45740-3), such filamentsare likely to be rapidly saturated with single-stranded binding proteinsand inactivated soon after initiation of the reaction. Thus forefficient RPA conditions that prevent inactivation of the reactioncomponents are key in establishing robust amplification.

We have predicted a potentially stable reaction composition using E.coli recA protein in the presence of ATP and the E. coli single-strandedbinding protein SSB, or the T4 uvsX protein in the presence of gp32. Wesuggested the presence of recO, recR, and possibly recF proteins (Bork,Cox and Inman EMBO J. 2001 Dec. 17; 20(24):7313-22), could lead to anenvironment in which pre-loaded recA filaments were stabilised, and inwhich recA could nucleate successfully onto SSB-bound oligonucleotides.A similar recombinase loading system has been described in otherorganisms including the recombination/replication/repair system ofbacteriophage T4. The T4 recombinase uvsX can be loaded ontosingle-stranded DNA coated with the T4 single-stranded DNA bindingprotein gp32 by the action of a third component, the uvsY protein(Morrical and Alberts J Biol Chem. 1990 Sep. 5; 265(25):15096-103).Interestingly a principal role of this recombination system in vivo isto permit recombination-dependant DNA synthesis by assemblingreplication components at recombination intermediates such as D-loops(Formosa and Alberts Cell. 1986 Dec. 5; 47(5):793-806). This process issimilar to what should happen in RPA driven from D-loops made by theinvasion of synthetic oligonucleotides. In addition to interactionsbetween the three components uvsX, uvsY, and gp32, there are alsointeractions between these components and the replication machinery suchas the polymerase, clamp loader, primase/helicase, and dda helicase(Reddy, Weitzel and Von Hippel, Proc Natl Acad Sci USA. 1993 Apr. 15;90(8):3211-5, Hacker and Alberts, J Biol Chem. 1992 Oct. 15;267(29):20674-81). Taken together these facts suggest that thecomponents of the T4 recombination/replication machinery would perhapsbe even more ideal for RPA than the E. coli equivalents.

In addition to the use of recombinase loading proteins, such as recO andrecR, or uvsY, there are other ways to create an appropriate balancebetween recombinase activity and the activity of single-stranded DNAbinding proteins. The DNA binding and/or cooperativity behaviour ofrecombinases and single-stranded DNA binding proteins can be modulatedby mutation. In addition, recombinases from different sources havedistinct properties (Eggler, Lusetti and Cox, J Biol Chem. 2003 May 2;278(18):16389-96. Epub 2003 Feb. 20, Villemain et al., J Biol Chem. 2000Oct. 6; 275(40):31496-504). This suggests that a range of recombinaseand single-strand DNA binding protein activities could be explored. Theuse of mutated proteins or proteins from different species, in a set ofoptimisation experiments could lead to the identification of an optimalratio of the competing recombinase and single-stranded bindingactivities. Ultimately the activities would be balanced such that DNAassociation/dissociation for the two DNA-binding species permitssufficient recombinase activity together with sufficient DNA meltingactivity of the single-strand DNA binding protein to perform itsnecessary functions also. In addition, reduction of noise due tomispriming may be achieved through the optimisation of such parametersas oligonucleotide sequence design, reaction buffer, the use of partlymodified oligonucleotides, the use of part duplex oligonucleotides orthe addition of other specific reaction components detailed below.

Here we provide the results of experiments that validate the RPA method.In particular, we provide a description and demonstration of reactioncompositions capable of supporting DNA amplification. We demonstratethat relatively short synthetic oligonucleotides can be used to targetspecific sequences and support initiation of DNA synthesis. We describethe requirements for particular types and concentrations of certainrecombinases, single-stranded binding proteins, ATP, and oligonucleotideconcentrations. We further describe the optimisation and modulation ofthe reaction environment, which supports an active and dynamicrecombination system with desired rate behaviour, through the inclusionof crowding agents (such as polyethylene glycols), recombinase loadingfactors and/or mutated proteins with altered biochemical activities. Weestablish that in the presence of distributive polymerases at least(e.g. the E. coli DNA polymerase I Klenow fragment), there aresubstantial improvements in amplification efficiency when the distancebetween the amplification priming sites is optimised. We establish thata balance between polymerase exonuclease activity and oligonucleotideprotecting agents must be employed to avoid non-specific degradation ofoligonucleotide primers. We show that amplification of sequencesembedded within linear (or relaxed) DNA substrates is relativelyinefficient (at least with very distributive polymerases such asKlenow), whereas amplification reactions directed toward the ends oflinear DNA substrates are most effective. We provide methods to preparetarget DNA to be more efficiently amplified in an lsRPA reaction,including the methods of thermal or chemical melting or restrictionenzyme digestion. We also provide evidence that the nature of thesingle-stranded binding protein is critical to establish efficient RPAreactions, and provide a rationale for this. Furthermore we suggestimprovements and novel approaches to reduce noise and optimiseamplification reactions performed at relatively low, or ambient,temperatures by the use of part-double-stranded oligonucleotides, oroligonucleotide wholly or partly lacking a phosphate backbone. We alsoprovide evidence that other enzymes and proteins involved in DNAmetabolism can influence RPA reactions, and some may be configured toimprove reaction efficiency and specificity. These includetopoisomerases, which can relax recombination/replication intermediatesand may aid targeting of embedded sequences, as well as helicases suchas T4 dda helicase or T4 gp41 which can improve the polymeraseinitiation and elongation efficiency, particularly if non-stranddisplacing polymerases are used. Finally we show that priA and theruvA/B helicases have activities that might be used to optimiseamplification efficiency.

Selection of RPA Reagents and Reaction Parameters

The details of leading strand RPA, leading and lagging strand RPA, andnested RPA were listed above. This section will describe the selectionof reagents and parameter for any of the three methods discussed above.

One benefit of RPA is that the amplified product of RPA is doublestranded DNA that could be used for other molecular biology procedures.Thus, RPA may be combined with other methods in molecular biology. Forexample, the starting material for RPA may be a PCR amplified fragment.Alternatively, the product of an RPA may be used for PCR.

If necessary, the RPA products in any of the methods of the inventionmay be purified. For example, in the nested RPA method, the amplifiedproduct may be purified after each RPA step before a subsequent RPAstep. Methods of purification of nucleic acids are known in the art andwould include, at least, phenol extraction, nucleic acid precipitation(e.g., with salt and ethanol), column chromatography (e.g., sizeexclusion, ionic column, affinity column and the like) or anycombination of these techniques.

As discussed, the primers used in RPA may be “double stranded” or“capable of forming double stranded structures.” These terms refer toDNA molecules that exist in a double stranded condition in a reactionsolution such as a RPA reaction solution or a PCR reaction solution. Thecomposition of a PCR solution is known. The composition of a RPAreaction is listed in this detailed description section and in theExamples.

The primers may have a single stranded region for hybridization to thetarget DNA in the presence of a recombinase agent. The single strandedregion may be, for example, about 10 bases about 15 bases, about 20bases, about 25 bases, about 30 bases, about 40 bases, and about 50bases. Even longer regions such as about 75 bases, about 100 bases,about 150 bases or more may be used but it is not necessary. The choiceof single stranded regions will depend on the complexity of the startingnucleic acid so that for example, a human genome may require a longerprimer while a plasmid may require a much shorter primer.

The two strands of nucleic acid in a double stranded DNA need not becompletely complementary. For example, the double-stranded region of adouble-stranded DNA may differ by up to 1% in sequence. That is, thesequence of two strands of nucleic acid may differ by one base in onehundred bases and would still exist in a double stranded condition insolution. Nucleic acids with 1% difference in their complementarysequence are contemplated as double stranded DNA for the purposes ofthis disclosure.

In addition, the target nucleic acid (i.e., the nucleic acid to beamplified by the RPA methods of the invention) may be partially doublestranded and partially single stranded. For example, nucleic acid in anyof the configuration of FIG. 5 would be suitable as a target nucleicacid of the invention. As discussed, the target nucleic acid may be RNA.RNA can be converted to double-stranded cDNA using known methods and thedouble-stranded cDNA may be used as the target nucleic acid. As shown ifFIG. 5, the template nucleic acid may have any combination of endsselected from 3′ overhang, 5′ overhang, or blunt ends.

The lsRPA method of the invention comprises at least the followingsteps. First, a recombinase agent is contacted to two nucleic acidprimers (referred to herein as a first and a second primer) to form twonucleoprotein primers (referred to herein as a first nucleoproteinprimer and a second nucleoprotein primer).

Second, the first and second nucleoprotein primers are contacted to thetemplate nucleic acid to form a double stranded structure at a firstportion of the first strand and a second double stranded structure at asecond portion of the second strand. The two primers are designed sothat when hybridized, they are pointed at each other as illustrated inFIG. 6A. Alternatively, primer 1 and primer 2 may hybridize differenttarget nucleic acids as illustrated in FIG. 6B.

Third, the nucleoprotein primers are extended at their 3′ ends togenerate a first and a second double stranded nucleic acid (FIG. 7A).Where the primers are hybridized to different target nucleic acids, theelongation of the primers will generate displaced strands (FIG. 7B). Inthis case, the two displaced strands that result from primer elongationmay hybridize and form a new double stranded template nucleic acid (FIG.7C).

Step two and three are repeated until the desired degree ofamplification is reached. The process is a dynamic process in thatprimer hybridization to the target nucleic acid and elongation areallowed to proceed continuously. One advantage of this invention is thatthe amplification is performed continuously without the need fortemperature cycling or enzyme addition after initiation of the reaction.

In an embodiment, steps two and three are repeated at least 5 times.Preferably, it is repeated at least 10 times. More preferably, it isrepeated at least 20 times, such as at least 30 times. Most preferably,the two steps are repeated at least 50 times. For multiple repetitionsof the amplification step (e.g., step 2 and 3) a RPA of the invention ispreferably started with a primer to target nucleic acid ration of atleast 100 to 1, preferably at least 300 to 1, and most preferably atleast 1000 to 1. That is, there are at least 100, 300 or 1000 copies ofthe primer per copy of a target nucleic acid.

In an optional step, after a sufficient round of amplification,additional components may be added to the reaction after a period oftime to enhance the overall amplification efficiency. In one embodiment,the additional components may be one or more of the following:recombinase agents, one or more primers, polymerase, and one or more ofthe additional agents (discussed in a separate section below).

In a preferred embodiment, a small fraction of a first RPA reaction isused as a supply of template DNA for subsequent rounds or RPAamplification. In this method, a first RPA amplification reaction isperformed on a target nucleic acid. After the first RPA reaction, asmall fraction of the total reaction is used as a substitute of thetarget nucleic acid for a subsequent round of RPA reaction. The fractionmay be, for example, less than about 10% of the first reaction.Preferably, the fraction may be less than about 5% of the firstreaction. More preferably, the fraction may be less than 2% of the firstreaction. Most preferably, the fraction may be less than 1% of theinitial reaction.

The primer used in RPA is preferably DNA although PNA, and RNA are alsosuitable for use as primers. It is noted that in fact, in DNAreplication, DNA polymerases elongate genomic DNA from RNA primers.

Synthetic oligonucleotides may serve as DNA primer and can be used assubstrates for formation of nucleoprotein filaments with RecA or itshomologues. Sequences as short as 15 nucleotides are capable oftargeting double-stranded DNA (Hsieh et al., 1992). Sucholigonucleotides can be synthesized according to standardphosphoroamidate chemistry, or otherwise. Modified bases and/or linkerbackbone chemistries may be desirable and functional in some cases.Additionally oligonucleotides may be modified at their ends, either 5′or 3′, with groups that serve various purposes e.g. fluorescent groups,quenchers, protecting (blocking) groups (reversible or not), magnetictags, proteins etc. In some cases single-stranded oligonucleotides maybe used for strand invasion, in others only partly single strandednucleic acids may be used, the 5′ stretch of sequence of an invadingnucleic acid being already hybridized to an oligonucleotide.

In another embodiment of the invention, the primers may comprise a 5′region that is not homologous to the target nucleic acid. It should benoted that the processes of the invention should be functional even ifthe primers are not completely complementary to the target nucleic acid.The primers may be noncomplementary by having additional sequences attheir 5′ end. These additional sequences may be, for example, thesequence for a restriction endonuclease recognition site or the sequencethat is complementary to a sequencing primer. The restrictionendonuclease recognition site may be useful for subsequent cleavage ofthe amplified sequence. The use of restriction endonuclease that cleavesnucleic acid outside the restriction endonuclease recognition site isalso contemplated. The sequence that is complementary for a sequencingprimer may allow rapid DNA sequencing of the amplified product usingcommercially available primers or commercially available sequencingapparatus.

Formation of nucleoprotein filaments can be performed by incubation ofthe primer (oligonucleotides) with RecA protein or its homologues in thepresence of ATP, and auxiliary proteins such as RecO, RecR and RecF, oruvsY in the case of T4 proteins. When incubated at 37° C. in RecA buffer(20 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 2 mM ATP, 2 mM DTT and 100 μg/mlBovine Serum Albumin), RecA will form helical filaments on ssDNA with 6protomers per turn. The DNA is located within the interior of theprotein helix. In the presence of dsDNA, the RecA/ssDNA nucleoproteinfilament can scan DNA at rates of at least 10₇ bp per hour. The mode ofscanning is unclear but it is at a speed (>10³ bp per second) that itmay involve only the initial few base pairs that can be easily accessedalong one face of the major groove. Successful binding may result in atransition to a triple-helical intermediate, which is then followed bystrand invasion and displacement to form a D-loop. Such joint moleculescan be formed under similar condition to those described above forformation of helical filaments, and hence in the presence of ssDNA, thehomologous dsDNA, RecA, ATP, auxiliary proteins and suitable buffer andtemperature conditions, joint molecules will form spontaneously. If ATPis used the assembly is reversible and will reach equilibrium, butRecA/ssDNA filaments can be stabilized, even in the presence of SSB, bythe auxiliary proteins RecO and RecR. Alternatively the T4 uvsX proteinmay be stabilized in the presence of uvsY protein. In the case ofthermostable proteins the temperature of incubation can be higher. If arenewable supply of ATP is required a standard ATP regeneration systemcan be included in the reaction.

DNA polymerases can use the free 3′-hydroxyl of the invading strand tocatalyze DNA synthesis by incorporation of new nucleotides. A number ofpolymerases can use the 3′-hydroxyl of the invading strand to catalyzesynthesis and simultaneously displace the other strand as synthesisoccurs. For example E. coli polymerase II or III can be used to extendinvaded D-loops (Morel et al., 1997). In addition, E. coli polymerase Vnormally used in SOS-lesion-targeted mutations in E. coli can be used(Pham et al., 2001). All of these polymerases can be rendered highlyprocessive through their interactions and co-operation with the.β.-dimer clamp, as well as single stranded DNA binding protein (SSB)and other components. Other polymerases from prokaryotes, viruses, andeukaryotes can also be used to extend the invading strand.

In another embodiment of the invention, the primer may be partiallydouble stranded, partially single stranded and with at least one singlestranded 3′ overhang. In this embodiment, the primer may comprise ainvading strand and a non-invading strand as shown in FIG. 8A. In thiscase, after the invading strand is hybridized to the target DNA andelongated, it serves as a target nucleic acid for a second primer asshown in FIG. 8B. The elongation of the second primer would displace thenoninvading strand as shown in FIG. 8C. In this embodiment, as thetarget nucleic acid is amplified, the non-invading strand of primer 1 isdisplaced. If both primer one and primer two are partly double strandedprimers, then the non-invading strands of both primer one and primer twowill accumulate in solution as the target nucleic acid is amplified.

In one embodiment of the invention, at least two of the primers in a RPAreaction are partially double stranded and partially single strandedeach generated by the hybridization of an invading strand and anon-invading oligonucleotide strand, which possess sequences ofsufficiently complementary that they form a double stranded region.Preferably, the two oligonucleotide strands are sufficientlycomplementary over the relevant region that they can form a doublestranded structure in RPA reaction conditions.

In an embodiment of the invention, the primers, includingsingle-stranded and partially double-stranded primers, are labeled witha detectable label. It should be noted that a fluorescence quencher isalso considered a detectable label. For example, the fluorescencequencher may be contacted to a fluorescent dye and the amount ofquenching is detected. The detectable label should be such that it doesnot interfere with an elongation reaction. Where the primer is partiallydouble stranded with an invading strand and a non-invading strand, thedetectable label should be attached in such a way so it would notinterfere with the elongation reaction of the invading strand. Thenon-invading strand of a partially double stranded primer is notelongated so there are no limitations on the labeling of thenon-invading strand with the sole exception being that the label on thenon-invading strand should not interfere with the elongation reaction ofthe invading strand. Labeled primers offer the advantage of a more rapiddetection of amplified product. In addition, the detection ofunincorporated label, that is, labeled oligonucleotides that have notbeen extended, will allow the monitoring of the status of the reaction.

Monitoring a RPA reaction may involve, for example, removing a fractionof an RPA reaction, isolating the unincorporated fraction, and detectingthe unincorporated primer. Since the size of an unincorporated primermay be less than 50 bp, less than 40 bp, less than 30 bp or less than 25bp, and the size of the amplified product may be greater than 1 Kb,greater than 2 Kb, greater than 5 Kb, or greater than 10 Kb, there is agreat size difference between the incorporated and unincorporatedprimer. The isolation of the unincorporated primer may be performedrapidly using size exclusion chromatography such as, for example, a spincolumn. If a primer is labeled, a monitor procedure comprising a spincolumn and a measurement (e.g., fluorescence or radioactivity) can beperformed in less than one minute. Another alternative for separatingelongated primers from unelongated primers involve the use of PAGE. Forexample, the elongated primer may be separated from the unelongatedprimer by gel electrophoresis in less than 5 minutes. Yet anotheralternative for separating elongated primers involves the use ofimmobilized oligonucleotides. For example oligonucleotides homologous tosequences found uniquely within the amplified DNA sequence can be usedto capture nucleic acids produced by primer elongation specifically.These capturing oligonucleotides can be immobilized on a chip, or othersubstrate. Capture of the elongated oligonucleotides by the capturingoligonucleotides can be performed by RecA protein mediated methods, orby traditional solution hybridizations if necessary.

In another embodiment of the invention, a double stranded primer may belabeled such that the separation of the two strands of the primer may bedetected. As discussed above, after multiple rounds of elongation, theinvading strand and the noninvading strands of a partially doublestranded primer is separated. After this separation, the non-invadingstrand does not participate in the RPA reaction. This characteristic maybe used to detect and monitor a RPA reaction in a number of ways.

In this application, the detectable label may be a fluorescent label oran enzyme and the label quencher (also referred to as the labelinhibitor) may be a fluorescence quencher or an enzyme inhibitor. Inthese cases, the label is detected by fluorescence or enzyme inhibition.The delectability of the label would be the fluorescence if afluorescent label is used or enzyme activity if an enzyme is used.

In the first method, the invading strand may be labeled with a label andthe non-invading strand may be labeled with a detectable label quencher.The label, in the proximity of the label quencher (label inhibitor) onthe partially double stranded primer would not be highly detectable.After RPA, the invading strand would be separated from the noninvadingstrand and thus, the label and the label quencher would be separated.The separation would cause the label to be more detectable. Thus,measuring the increases in the amount of detectable label may monitorRPA reactions.

The second method is similar to the first method except that theinvading strand is modified with a label quencher while the noninvadingstrand is modified with a label. Then RPA is allowed to proceed with theresult (same as method 1) of the label being separated from the labelquencher. Thus, the overall delectability of the label would increase.

The third method involves labeling the noninvading strand of one doublestranded primer with a label. In addition, the noninvading strand of asecond double stranded primer is labeled with a label quencher. The twonon-invading stands are designed to be complementary to each other. Inthis configuration, the RPA reaction is initially fluorescent. As theRPA reaction progresses, the two noninvading strands are displaced intosolution and they hybridize to each other because they are designed tobe complementary. As they hybridize, the label and the label quencherare brought into proximity to each other and the fluorescence of thereaction is decreased. The progress of the RPA reaction may be measuredby monitoring the decrease in label detectability.

In a fourth method, the noninvading strands of a first and second doublestranded primers are labeled with a first label and a second label. Thetwo noninvading strands are also designed to be complementary to eachother. As in the third method, after RPA, the two noninvading strandsare hybridized to each other and the proximity of the two labels will bea reflection of the progress of the RPA reaction. The proximity of thetwo labels may be determined, for example, by direct observation or byisolation of the non-invading strands. As discussed above, isolation ofprimers and other small nucleic acids can be accomplished by sizeexclusion columns (including spin columns) or by gel electrophoresis.

In another embodiment of the invention, the non-invading strand of oneor both of the primers is homologous to a second region of nucleic acidsuch that the primer can hybridize to and primer DNA synthesis at thesecond region of nucleic acid. Using this method, a second RPA reactionusing the noninvading stand from the primer of a first RPA may bestarted. The product of the second RPA may be monitored to determine theprogress of the first RPA.

In yet another embodiment of the invention, the non-invading strand isdetected by a biosensor specific for the sequence of the non-invadingstrand. For example, the biosensor may be a surface with a nucleic acidsequence complementary to the non-invading strand. The biosensor maymonitor a characteristic that results from the binding of thenon-invading strand. The characteristic may be a detectable label.

Suitable detectable labels for any of the methods of the inventioninclude enzymes, enzyme substrates, coenzymes, enzyme inhibitors,fluorescent markers, chromophores, luminescent markers, radioisotopes(including radionucleotides), and one member of a binding pair. Morespecific examples include fluorescein, phycobiliprotein, tetraethylrhodamine, and β-gal. Bind pairs may include biotin/avidin,biotin/strepavidin, antigen/antibody, ligand/receptor, and analogs andmutants of the binding pairs.

The recombinase agent of the invention may be RecA, uvsX, RadA, RadB,Rad 51 or a functional analog or homologues of these proteins. Ifdesired, the recombinase may be a temperature-sensitive (referred toherein as “ts”) recombinase agent. If a ts recombinase is used, the RPAreaction may be started at one temperature (the permissive temperature)and terminated at another temperature (the non permissive temperature).Combinations of permissive temperatures may be, for example 25° C./30°C., 30° C./37° C., 37° ° C./42° C. and the like. In a preferredembodiment, the ts protein is reversible. A reversible ts protein'sactivity is restored when it is shifted from the nonpermissivetemperature to the permissive temperature.

In a preferred embodiment, the RPA is performed in the presences of ATP,an ATP analog, or another nucleoside triphosphate. The ATP analog maybe, for example, ATPγS, dATP, ddATP, or another nucleoside triphosphateanalog such as UTP.

Other useful reagents that may be added to an RPA reaction includenucleotide triphosphates (i.e., dNTPs such as dATP, dTTP, dCTP, dGTP andderivatives and analogs thereof) and a DNA polymerase. Other usefulreagents useful for leading/lagging RPA include NTPs (ATP, GTP, CTP, UTPand derivatives and analogs thereof). One advantage of the RPA reactionis that there is no limit on the type of polymerase used. For example,both eukaryotic and prokaryotic polymerases can be used. Prokaryoticpolymerase include, at least, E. coli pol I, E. coli pol II, E. coli polIII, E. coli pol IV and E. coli poly. Eukaryotic polymerases include,for example, multiprotein polymerase complexes selected from the groupconsisting of pol-α, pol-β, pol-δ, and pol-ε.

In another embodiment of the invention, the RPA process is performed inthe presence of an accessory component to improve polymeraseprocessivity or fidelity. Both eukaryotic and prokaryotic accessorycomponents may be used. Preferably, the accessory component is anaccessory protein is from E. coli. Useful accessory proteins includesingle-strand binding protein, helicase, topoisomerase, and resolvase.Other useful accessory proteins include a sliding clamp selected fromthe group consisting of an E. coli β-dimer sliding clamp, a eukaryoticPCNA sliding clamp and a T4 sliding clamp gp45. Other accessorycomponents include a DNA Polymerase III holoenzyme complex consisting ofβ-Clamp, DnaX Clamp Loader, and the Polymerase Core Complex. Still otheraccessory components include RuvA, RuvB, RuvC, and RecG. The propertiesendowed by the use of additional components will likely enable theamplification of large DNAs not previously successfully targeted bycurrent methods such as PCR.

In another embodiment, the RPA is performed in the presence of agentsused to stabilize recombinase/ssDNA nucleoprotein filaments. Forexample, the agent may be RecR, RecO, RecF, or a combination of theseproteins, or T4 uvsY protein if T4 components are used. Molecularcrowding agents may also be employed to modulate biochemicalinteractions in a favourable manner. Other useful agents include PriA,PriB, DnaT, DnaB, DnaC, and DnaG.

One benefit of the present invention is that the RPA reaction may beperformed at reduced temperatures compared to a PCR reaction. Forexample, the RPA process may be performed between 20° C. and 50° C.Preferably, the RPA process is performed at less than 45° C. Morepreferably, the RPA process may be performed at less than 40° C. Evenmore preferably, the RPA process may be performed at less than 35° C.Most preferably, the RPA process may be performed at less than 30° C.One of the reasons that the RPA process can be performed at thesereduced temperatures is because RPA may be performed without temperatureinduced melting of the template nucleic acid. Further, unlike PCR,absolute temperature control is not required and the temperature canfluctuate without adversely affecting RPA. For example, the amount offluctuation may be anywhere within the temperatures specified above. Thetemperature necessary for melting of double stranded DNA also contributeto premature enzyme inactivation, a disadvantage absent in the methodsof this invention.

RPA may be performed to test for the presences or absences of agenotype. The genotype tested may be associated with a disease or apredisposition to a disease. Alternatively, the genotype may beassociated with a normal phenotype or a phenotype that confers specialresistance to a disease. The genotype as disclosed above may be anystandard genetic variant such as a point mutation, a deletion, aninsertion, an inversion, a frameshift mutation, a crossover event, orthe presence or absences of multiple copies of a genetic sequence (e.g.,the presences of minichromosomes).

One method of detecting a genotype is to detect the distance between aprimer pair in an RPA reaction. The distance between a primer pair isreflected by the size of the amplified sequence. In that method, the twoprimers are selected such that it spans a target region such as, forexample, a gene. Then RPA is performed using the primer pair and the RPAproduct is analyzed. The analysis may involve determining the size orsequence of the amplified product. Methods of determining the size of aDNA sequence, including at least techniques such as agarose gels, PAGEgels, mass spectroscopy, pulsed field gels, gene chips, sucrosesedimentation and the like are known. There are many DNA sequencingmethods and their variants, such as the Sanger sequencing using dideoxytermination and denaturing gel electrophoresis (Sanger, F., Nichlen, S.& Coulson, A. R. Proc. Natl. Acad. Sci. U.S.A. 75, 5463-5467 (1977)),Maxam-Gilber sequencing using chemical cleavage and denaturing gelelectrophoresis (Maxam, A. M. & Gilbert, W. Proc Natl Acad Sci USA 74,560-564 (1977)), pyrosequencing detection pyrophosphate (PPi) releasedduring the DNA polymerase reaction (Ronaghi, M., Uhlen, M. & Nyren, P.Science 281, 363, 365 (1998)), and sequencing by hybridization (SBH)using oligonucleotides (Lysov, I., Florent'ev, V. L., Khorlin, A. A.,Khrapko, K. R. & Shik, V. V. Dokl Akad Nauk SSSR 303, 1508-1511 (1988);Bains W. & Smith G. C. J. Theor. Biol 135, 303-307 (1988); Dmanac, R.,Labat, I., Brukner, I. & Crkvenjakov, R. Genomics 4, 114-128 (1989);Khrapko, K. R., Lysov, Y., Khorlyn, A. A., Shick, V. V., Florentiev, V.L. & Mirzabekov, A. D. FEBS Lett 256. 118-122 (1989); Pevzner P. A. JBiomol Struct Dyn 7, 63-73 (1989); Southern, E. M., Maskos, U. & Elder,J. K. Genomics 13, 1008-1017 (1992)).

One method of detecting a genotype is to use primers that are specificfor a particular genotype. For example, a primer may be designed toefficiently amplified one genotype but inefficiently or not amplifyanother genotype at all. In an embodiment, the primer may comprise a 3′sequence that is complementary to one genotype (e.g., a genetic diseasegenotype) but not to another genotype (e.g., a normal genotype).

The genotype to be determined may be indicative of a disease such as,for example, the presence of an activated oncogene; the presence of thegene for Huntington's disease or the absence of an anti-oncogene.

The 3′ bases of the primers are especially important in determining thespecificity and efficiency of an RPA reaction. A primer may be designedso that the 3′ base is complementary to one genotype and notcomplementary to another genotype. This will allow efficient RPA of onegenotype and an inefficient RPA (if any) of the second genotype. It isnoted that the method is effective if only one primer of the primer paircan differentiate between different phenotypes (by having differentefficiencies of amplification). In a preferred embodiment, both primersin an RPA reaction can differentiate between different genotypes. Inthis above example, the primers are complementary to one genotype andare not complementary to a second genotype by one base at its 3′ end. Ina preferred embodiment, the primer is not complementary to the secondgenotype by at least one base at its 3′ end. Preferably, the primer isnot complementary to the second genotype by at least 2, 3, 4, 5, 6, 7,8, 9, or 10 bases at its 3′ end. Most preferably, the primer iscompletely non-complementary or cannot hybridize to the second genotypewhile it can hybridize to the first genotype.

In some of the methods discussed, the presence or absence of anamplified product provides the indication of the presence or absence ofa genotype. In these cases, the RPA reaction may be monitored by themethods discussed throughout the specification.

In a preferred embodiment, an RPA reaction for genotyping will amplify asequence regardless of the genotype of the patient. However, thegenotype of a patient will alter a characteristic of the amplifiedsequence. For example, the amplified sequence may be a different size,or sequence for one genotype than for another genotype. In that way, theRPA reaction will contain an internal control to indicate that theamplification reaction was performed successfully. Naturally, a methodof RPA, which includes one or more additional pairs of primers ascontrols for the performance of the RPA reaction, is also envisioned.

In another embodiment, an RPA reaction may be used to determine thepresence or absences of a nucleic acid molecule. The nucleic acidmolecule may be from any organism. For example, the microbialcomposition of a sample may be determined by using a battery of RPAreactions directed to the nucleic acid of different microbes. RPA isespecially useful for the detection of microbes. In one embodiment, thepathogens are selected from viruses, bacteria, parasites, and fungi. Infurther embodiments, the pathogens are viruses selected from influenza,rubella, varicella-zoster, hepatitis A, hepatitis B, other hepatitisviruses, herpes simplex, polio, smallpox, human immunodeficiency virus,vaccinia, rabies, Epstein Barr, retroviruses, and rhinoviruses. Inanother embodiment, the pathogens are bacteria selected from Escherichiacoli, Mycobacterium tuberculosis, Salmonella, Chlamydia andStreptococcus. In yet a further embodiment, the pathogens are parasitesselected from Plasmodium, Trypanosoma, Toxoplasma gondii, andOnchocerca. However, it is not intended that the present invention belimited to the specific genera and/or species listed above.

Here we present data that help to define reaction conditions that permitefficient amplification of DNA by RPA.

Single-Stranded DNA Binding Protein

Single-stranded DNA binding proteins are required for RPA reactions.These proteins bind to single-stranded DNA, melt secondary structure,facilitate outgoing strand displacement, and suppress branch migration.In RPA their activity is required during several distinct phases. Wehave investigated the activities of two single-stranded DNA bindingproteins, E. coli SSB and bacteriophages T4 gp32. The T4 gp32 has provento be most useful in our hands. Furthermore we have generated a numberof distinct forms of this protein by including hexahistidine (His)peptide tags at the N or C termini, as well as investigating severalpreviously described point mutations. Activities of gp32 variants aredepicted schematically in FIG. 21.

Variant Forms of gp32

T4 gp32 protein possesses several features that are of potential utilityto RPA reactions. Foremost gp32 has a relatively small DNA binding site(8-10 nucleotides), displays similar binding properties under a widerange of salt concentrations, and displays high (unlimited)cooperativity between monomers (Scheerhagen et al., J Biomol Struct Dyn.1986 April; 3(5):887-98; Kuil et al., Biophys Chem. 1988 December;32(2-3):211-27). In contrast, E. coli SSB protein has several distinctDNA binding modes that vary with salt concentration all of which possessrelatively large DNA binding sites (32, 56 or 65 nucleotides)(Ferrari etal., J Mol Biol. 1994 Feb. 11; 236(1):106-23) and there is complexcooperativity behaviour (Lohman and Ferrari, Annu Rev Biochem. 1994;63:527-70). Because the initial size of the outgoing strand is smallwhen synthetic oligonucleotides are employed, we reasoned that theproperties of the gp32 protein would be optimal for RPA. We expressedand purified gp32 possessing an N-terminal His tag (gp32(N)). In initialexperiments we found gp32(N) to function at least as well as the E. coliSSB protein, even when combined in a heterologous system with the E.coli recA recombinase (FIG. 12). This was surprising result as gp32 isreported to display extremely high cooperativity between monomericsubunits and it seemed unlikely that recA would be able to competeeffectively for oligonucleotide binding in its presence. When wecompared the behaviour of gp32(N) to untagged gp32, however, wediscovered that the two proteins did not behave equivalently. As theN-terminal His tag is directly adjacent to the ‘B’ domain of the gp32protein, which is required for cooperativity between monomers, wereasoned that gp32(N) must have attenuated cooperativity. We thereforegenerated a gp32 protein possessing a C-terminal His tag (gp32(C)), aswell as point mutant forms of gp32(C) in accordance with previouslypublished mutants having a lysine to alanine change at position 3 (K3 toA), or an arginine either glutamine (R4 to Q) or threonine at position 4(R4 to T) (FIG. 21). These three point mutant proteins exhibitprogressively less cooperativity (FIG. 22) (Villemain, et al. J BiolChem. 2000 Oct. 6; 275(40):31496-504). We tested the capacity of theseproteins at two different concentrations to support invasion/extensionreactions on a linearized template in combination with the bacteriophageT4 uvsX protein and the Klenow fragment of E. coli DNA polymerase I(FIG. 22). Firstly we note that gp32(C) yields much less product thanother gp32 variants at either concentration (FIG. 22 compare withgp32(N)) and that the products are almost exclusively full-length, incontrast to gp32(N). When we compare these results to those obtainedwith the point mutant allelic series we note that the gp32(N) proteinmost closely resembles the profile obtained with gp32(C)R4 to T, whichhas been reported to be significantly attenuated in cooperativity. Thissuggests that the N terminal His tag of gp32(N) interferes with thefunction of the B domain in a manner similar to the point mutations.

There are several other relevant observations. Firstly, the proteinsbelieved to demonstrate the highest cooperativity, i.e.gp32(C)>gp32(C)K3A seem to produce less product. Secondly, for gp32(C)and gp32(C)K3A, more amplified product is generated when lesssingle-stranded binding protein is used, in contrast to gp32(N) andgp32(C)R4T which generate more product in run-on assays when moreproteins is used. Taken together these observations suggest anexplanation. If the gp32 species is progressively more cooperative thenit will form progressively more stable filaments on the oligonucleotidesand make it progressively more difficult for recombinase to load.Consequently when the most cooperative gp32 species are employed thereis a significant limitation on the availability of recombinase-loadedfilaments. If the concentration of gp32 is raised, recombinase loadingis further suppressed by gp32 single-stranded DNA binding activity.Consistent with this, as the cooperativity of the gp32 is progressivelydecreased the amount of amplified product increases. This is consistentwith a substantial increase in recombinase-coated filament formation. Asrelatively un-cooperative gp32 monomers are less likely to coatoligonucleotides and permit the recombinase, uvsX, to seed instead. Wenote that in the case of gp32(N) and gp32(C)R4T, more product isgenerated with increased gp32(N) or gp32(C)R4T in DNA run-on assays.This contrasts results using the more cooperative gp32 variants. Onepossibility is that during and following the strand exchange reactiongp32 is required to stabilise the recombination and synthesisintermediates. This may happen because gp32 cooperativity is soattenuated that it may no longer participate in those aspects of thereaction, or because the recombinase out-titrates gp32 and halts thereaction. The contrasting results of amplification versus run-on assayssuggest that the optimal amount of cooperativity may be less than thatpossessed by the most cooperative gp32 variants. Thus for RPA mutantgp32 variants, with attenuated cooperativity, may be the mostappropriate as these will permit higher levels of recombinase loading.Attenuation of gp32 cooperativity must, however, be balanced againstnoise generated by mispriming events as may be encountered inenvironments with less gp32 activity.

Finally, there is a substantial difference in the quantity of productgenerated in RPA using 7.5 μg versus 15 μg of gp32(C)K3A. The 7.5 μglevel more closely resembles results with more weakly cooperativemutants. Most likely in some cases during the course of the reaction,which was held at 37° C. for 2 hours, the amount of single-strandedrun-on product increases to the point that gp32 no longer effectivelysaturates single-stranded DNA in the reaction. In such a situation twothings would occur. First there would be a rapid increase in the numberof homology-searching filaments leading to a significant rate increasein invasion. Then the lack of gp32 to stabilise outgoing strands wouldlead to many partial extensions by Klenow not being stabilised, andpossibly a greater rate of bubble migration separating the new strandfrom the template. Hence many more synthesised strands would not achievefull length.

A Model of gp32 Function in Multiple Invasion/Extension Reactions

We had earlier noted that in a heterologous system involving recA andgp32(N), polyethylene glycol was required to permit more than one cycleof invasion and extension from a given template. Here we describe amodel to rationalise these observations and explain why targeting DNAends multiple times requires specific types of single-stranded DNAbinding protein. It is clear gp32 activity is required to stabilise theoutgoing strand during the strand exchange process. If this does notoccur then as the recombinase disassembles the outgoing strandre-hybridises with its complementary strand and displaces the invadingoligonucleotide. Following strand exchange the outgoing strand can existin one of two states, which likely place different demands on thesingle-stranded DNA binding protein. These two states arise as aconsequence of the relationship between the single-stranded searchingDNA and the duplex target DNA. If the recombination event can extend toan end of a linear duplex DNA, and it is possible for the outgoingstrand to be completely removed from its complement at one end, then theoutgoing strand is un-constrained at one end. Under this un-constrainedcondition the new duplex involving the incoming DNA strand and itscomplement is able to rewind to form B form DNA. This is necessarybecause during the pairing reaction the target DNAs are under wound bythe activity of the invading recombinase filament. Un-constrainedoutgoing strands can readily bind single-stranded DNA binding proteins,which will prevent branch migration from occurring and allowing theinvading strand to be removed.

Alternatively if the recombination event does not extend to the end ofthe target duplex, as would occur if an embedded sequence were targeted,then the outgoing strand is topologically constrained because it isphysically joined to the complementary strand upstream and downstream ofthe recombining region. Such intermediates are highly unstable becausethe newly formed duplex cannot rewind without making the outgoing strandalso wind around it. And since the outgoing strand is constrained atboth ends this is energetically unstable, and the new hybrid is placedunder considerable strain. Consequently a far higher demand is placed onthe activity of single-stranded DNA binding proteins in this contextbecause there is substantial drive to eject the incoming strand andrewind the original duplex (FIG. 6).

In our efforts to establish effective conditions for repeated strandinvasion and extension of linear DNA targets we have noted that onlyoligonucleotides targeted to the ends of linear sequences are readilyextended to full template length, at least when using a distributivepolymerase such as the Klenow fragment of E. coli DNA polymerase I. Wehave found that under certain conditions, for example when using recAwith gp32(N) or E. coli SSB, only one round of invasion and extensioncan readily occur on each target template. These observations may beunderstood by considering the outcome of invasion events occurring underseveral different circumstances leading to either fully releasedoutgoing strands (plectonemic joints) or topologically constrainedintermediates (paranemic joints). Finally, we have identified certainother conditions that are permissive for multiple rounds of invasion andextension from a single template, a situation ideally suited to the RPAreaction. We propose a model based on our own data to consolidate theseobservations, and forms a framework around which optimisation of the RPAreaction can be designed. This model takes into account the behaviour ofgp32 protein under different reaction environments and justifies gp32behaviours with the effects of other reaction components (FIG. 29).

The model schematically describes the nature and outcome ofrecombination events between the end of a target duplex and an invadingoligonucleotide that initially possesses a 5′ overhang relative to thetarget. This situation typifies the experimental circumstance we havestudied. Despite this starting situation all but the initial cycleinvolves the invading oligonucleotides having their 5′ extreme flush tothe 5′ extreme of the target duplex DNA. This is the case because thetarget DNA complementary strand possesses a 3′-end that can be extendedduring the first round to copy the overhang region of the invadingprimer, which we term backfire synthesis. Experiments performed withgp32(N) protein (in the absence of PEG), suggest that once the target isflush at the 5′-end to the oligonucleotide i.e. after the first round,then subsequent invasion/extension cycles are very inefficient or do notoccur at all. Early experiments showed only a roughly equimolar quantityof run-on product to start template. How does this occur?

We believe that this block to re-invasion/extension arises becauseinvading oligonucleotides are rarely fully coated by recombinase. Thuscomplete exchange to the 5′ flush end of a target will also occur veryrarely and corresponding outgoing strands will remain in a constrainedstate (FIG. 13). The exchange is initially incomplete and theintermediate is unstable due to an inability to allow the new duplex torelax into a B-DNA helix in the presence of the topologicallyconstrained outgoing strand. Such unstable intermediates will have atendency to rewind the original duplex and eject the invading strand asthe recombinase disassembles.

We found, however, that if a crowding agent such as polyethylene glycolis included in the presence of gp32(N), subsequent invasion/extensionoccurs. One possibility is that under these conditions unstableintermediates are temporarily stabilised by gp32(N), such thatelongation can occur. It is possible that polyethylene glycol acts topartly rescue the poor cooperativity of the compromised gp32(N) allowingit to stabilise these otherwise unstable intermediates. This conclusionis supported both by our data showing that N-terminally his-tagged gp32is cooperatively attenuated, and the known capacity of crowding agentsto enhance the effectiveness of interactions between molecules, thuspartly making up for the poorer interaction between monomers. Weinitially believed that this experiment might be best explained bysuggesting that recA filaments were more abundant in the presence ofPEG, as previously reported elsewhere (Layery P E, Kowalczykowski S C. JBiol Chem. 1992 May 5; 267(13):9307-14), however we feel that this doesnot adequately account for the observed switch from a dead-end only oneround of invasion to productive multiple rounds of invasion/extension.

The difference between PEG stimulation of full-length multiple run-onsat end-directed targets versus the far poorer activity at truly embeddedtargets (FIG. 15), suggests that the temporary stabilisation of anintermediate, as suggested above, is insufficient alone to generatesubstantial elongation. If this were the case for run-on assays atembedded targets it should work as well in reinvasion at end-directedtargets, but this is not the case (FIG. 15). Alternatively thedifference between efficiencies may be explained by supposing that gp32temporarily stabilises an intermediate in which the 5′ extent of theincoming oligonucleotide is not paired but free, and possibly evencoated with gp32 dependent on the length. Provided that this unexchangedsegment is not coated with gp32, or that the gp32 can readilydissociate, as would be the case without cooperativity, then the 5′-mostportion of the oligonucleotide could become paired via rapid branchmigration (FIG. 29, scenario 1). In fact there is a difference betweentruly embedded sequences and re-invasion at DNA ends (FIG. 15). Forembedded targets, secondary branch migration leading to unconstrainedrelease of the outgoing strand can never occur because the ends are toodistant.

We would therefore conclude that the small DNA binding size, and highcooperativity between subunits permits gp32 proteins permits multipleinvasion and extension reactions by stabilising the constrainedparanemic joint structures for a sufficient time. There are variouspossible outcomes (FIG. 29). Either the last small section ofoligonucleotide at the 5′ end that was not initially exchanged becomeshybridised through a branch migration event, or disassembly of gp32 onthe outgoing strand permits the invading/extending oligonucleotide to belost through branch migration. Alternatively, loading of recombinaseonto the outgoing strand and re-invasion ejects the incoming strand(bubble migration). If hybridisation via branch migration occurs then anunconstrained structure arises that can be readily stabilised andextended. If either gp32 disassembly or bubble migration occur thenthere is a substantial risk that the new extending strand will be lostbefore it is fully extended. If a single stranded DNA binding proteinwith a large binding site, like E. coli SSB, or one with poorcooperativity, like. gp32(N), is used in the absence of PEG, then notemporary stabilisation occurs and the invading oligonucleotide isejected without being extended.

Consequently based on this model, and the earlier conclusions about thefrequency of recombinase-loaded filaments in the presence of various g32forms, we conclude that a balance between the activities of recombinasesand gp32 molecules must be struck that best meets the variousrequirements of the amplification reactions. We can summarise the needsand effects of single-stranded DNA binding proteins in different phasesof the RPA reaction as follows.

Phase 1

Single-stranded DNA binding proteins help to prepare single-strandedDNAs for recombinase loading by melting secondary structure so thatrecombinase loading can occur consistently. Thus the melting activity ofsingle-stranded DNA binding proteins is desirable and also plays a partin silencing non-specific annealing of primers. Despite this, however,excessive levels of protein and excessive cooperativity cansignificantly reduce number of recombinase-loaded filaments availablefor invasion.

Phase 2

Single-stranded DNA binding proteins collect the outgoing strand andprevent spontaneous ejection of the incoming oligonucleotide as therecombinase disassembles. The instability of paranemic joints means thatinvasions occurring on embedded sequences, including the case thatoligonucleotide ends are flush to the duplex ends (as would occur duringmost cycles of an amplification), means that significant cooperativeactivity may be required for many situations. In general this phase ofthe reaction will benefit from a surplus of highly cooperativesingle-stranded DNA binding proteins.

Phase 3

Single-stranded DNA binding proteins bind to the displaced strand thatforms during DNA synthesis. As in phase 2 this displaced strand may beunconstrained, or topologically constrained, and these two circumstancesplace different demands on the single-stranded DNA binding protein.

Phase 4

In certain configuration of the RPA reaction the displacedsingle-stranded outgoing strand must hybridise to a partneroligonucleotide to permit subsequent generation of a new duplex. Manysingle-stranded DNA binding proteins prevent complementary strands fromannealing, however T4 gp32 protein aids re-annealing of complementaryDNA. Therefore bacteriophage T4 gp32 protein is an ideal protein forthis phase.

Oligonucleotide Length:

There is little published evidence to support how effectively recA, orother recombinases, might be used with relatively the short syntheticoligonucleotide primers used for RPA. It is also unclear whether astable reaction environment can be generated in which such short DNAoligonucleotides remain actively loaded with recombinase. Most studiesperformed with recA utilise comparatively large substrates such assingle-stranded and double-stranded forms of the bacteriophage M13genome (many thousands of residues) as donor and acceptor DNAs.Experimental assays for recombinase activity often consist of theformation of intermediate, or completed, recombination events measuredby electrophoretic migration or electron microscopy (Harris L D,Griffith J. J Biol Chem. 1987 Jul. 5; 262(19):9285-92). A fewexperiments have been described using short oligonucleotides. Sequencesas short as 15 nucleotides have been shown to assemble functionalhomology-searching complexes with recA in the presence of thenon-hydrolysable cofactor analogue ATP-γ-S, but investigations combiningshort oligonucleotides and ATP are ambiguous (Hsieh P, Camerini-Otero CS, Camerini-Otero R D. Proc Natl Acad Sci USA. 1992 Jul. 15;89(14):6492-6). The homology-searching function of recombinases is notnecessarily sufficient to complete strand exchange and to release fromthe invasion complex to allow access by other DNA metabolising proteinssuch as polymerases. Indeed, studies have shown that recA showstransitions between low ATP hydrolysis rate (a useful indicator ofcombined DNA binding activity and functional recombinase activity) andhigh hydrolysis rate at oligonucleotide lengths substantially longerthan the 15 nucleotides required for searching. Furthermore the type ofnucleotide cofactor seems to influence on the length at which suchhydrolysis transitions occur (Katz F S, Bryant F R. Biochemistry. 2001Sep. 18; 40(37):11082-9). The bacteriophage T4 recombinase uvsX has alsobeen shown to exhibit variable properties on short oligonucleotides, andshows some sensitivity to the base composition (Formosa T, Alberts B M.J Biol Chem. 1986 May 5; 261(13):6107-18). Despite this, both uvsX andrecA are capable of performing recombination events with single-strandedsubstrates of roughly 30 base pairs or more in the presence ofhydrolysable nucleotides like ATP, suggesting that the use of such shortsynthetic targeting oligonucleotides is reasonable (Salinas F, Jiang H,Kodadek T. J Biol Chem. 1995 Mar. 10; 270(10):5181-6; Formosa T, AlbertsB M. J Biol Chem. 1986 May 5; 261(13):6107-18).

An oligonucleotide bearing 33 residues of homology with the end of alinearized DNA target can form a pairing intermediate capable ofelongation by the Klenow fragment of E. coli (FIG. 9). This experimentand others demonstrate that homology lengths as short as 33 nucleotidesare sufficient to direct recombinase/ssDNA filaments to appropriatetargets in the presence of ATP and to permit complete strand exchange.Similar results are found when using the bacteriophage T4 uvsX protein.

In addition to a requirement for a minimal oligonucleotide length theremay be a progressive loss of invasion/extension efficiency if theoligonucleotide is extended significantly beyond the minimal lengthrequired for recombination, at least when distributive polymerases areused. One possibility lies in the nature of recombinase/ssDNA filaments.Filaments coated with recombinases have varying 5′ limits to theircoating, as recombinases seed randomly and then extend the filament in a5′-3′ direction, there will be a distribution of 5′ extents of coating.If less than roughly 25-30 nucleotides are coated then littlerecombination can occur because there is insufficient nucleoproteinfilament for strand exchange. If more than this is coated it ispotentially beneficial from the point of view of recombination, but ifgreater than 10-20 residues is added beyond the minimal length requiredfor exchange there is a possibility that progressively more activefilaments will posses recombinase of a sufficient length of DNA topermit exchange, but retain sufficient 5′ uncoated DNA for gp32 to bind,which through cooperative binding of the 5′ extreme could inhibit thebranch migration phase and prevent the outgoing strand from beingun-constrained. Consistent with this notion, we note that stimulation ofmultiple invasion events was less apparent with the oligonucleotide thelonger oligonucleotide primer Tester1 bio compared to Tester3 bio (FIG.15). The only difference between these oligonucleotides is that thefirst has 25 additional overhanging nucleotides beyond the initial 33residues of homology, while the second has only 15 additional residues.Regardless of the explanation this experimental observation argues thatan optimal maximal length may exist.

There are other reasons to suspect shorter oligonucleotides will be bestfor efficient RPA. At the relatively low temperatures used in RPAreactions there is a substantial increase in stable secondary structuresof oligonucleotides as well as a greater probability of inappropriatehybridisation between primer pairs. Despite an overall excess ofsingle-stranded DNA binding proteins, the dynamic nature of the reactionsuggested by the instability of nucleoprotein filaments formed withrecombinases in the presence of ATP means that there is likely to be aconstant cycling of proteins on and off oligonucleotides, and asteady-state concentration of uncoated, unprotected, oligonucleotides.Consequently the use of short oligonucleotides should reduce thelikelihood of undesirable intra- and intermolecular interactions.

Our data indicate that optimal length of oligonucleotide lay between 30nucleotides and 50 nucleotides, and that progressively largeroligonucleotides can decrease the rate of invasion/extension. It may,however, be desirable to extend the length of the oligonucleotide toaccommodate a duplex region in the 3′ or 5′ region of the searchingoligonucleotide. Thus, in one aspect of the invention, the preferredprimer length is between about 30 to about 50 bases. Examples of primerssizes that would fit at least one of these criteria includes primers ofbetween 30 to 45 bases, between 30 to 40 bases, between 30 to 35 bases,between 35 to 40 bases, between 40 to 45 bases, and between 45 to 50bases. While the above-referenced primer sizes are preferred, arecombinase and/or single-stranded binding protein with an optimumprimer length of less than 30 bases is also possible and envisioned.

Oligonucleotide composition, sequence and single-stranded/duplexcharacter:

1 Composition and Sequence:

Software to design oligonucleotides for use in vitro DNA synthesisreactions is well established, particularly for use in PCR. Theconsiderations for the RPA method are similar and include theoptimisation of the melting temperature of the oligonucleotide,avoidance of hairpin formation within an oligonucleotide and selectionagainst complementarity with other oligonucleotides present in a givenreaction. As RPA is performed at relatively low temperatures suchconsiderations are potentially more important. We have observed theaccumulation of extended primer products in RPA reactions, which areapparently template-independent and dependent on the combination ofprimers used. The sizes of the aberrant products generated suggest theyare primer dimers, or the consequence of self-priming of a singleoligonucleotide. These undesirable primer artifacts are well known forother methods such as PCR. It is therefore important to designoligonucleotide primer pairs to avoid undesirable side reactions. Wehave observed that oligonucleotides capable of forming hairpins canerroneously self-prime in an RPA reaction.

Besides optimising oligonucleotide sequence design there are additionalapproaches to reduce or eliminate primer dimer formation. We haveobserved that reaction noise can be significantly reduced by utilisingpolymerases lacking 3′-5′ exonuclease activity. This suggests misprimingmay result from oligonucleotides that have been shortened by the 3′-5′exonuclease activity of polymerases. Consequently 3′-5′ exonucleaseediting activity, pyrophosphorylysis, or any other similar editingactivity can be a source of noise. In addition to using polymeraseslacking exonuclease activity and the removal of pyrophosphate withpyrophosphatase, use of synthetic oligonucleotides with anon-hydrolysable backbone at the ultimate and/or penultimate link may bebeneficial to reduce reaction noise. Alternative backbones could beselected from the considerable range of chemistries available such asphosphorothiorate, morpholino, locked nucleic acid, or peptide nucleicacid.

2) Single-Stranded/Duplex Character:

Deterring aberrant extension of oligonucleotide 3′ ends may also beachieved by designing, and including, short competitor oligonucleotidesthat can efficiently compete for the formation of hybrids with the 3′region of the targeting oligonucleotides. Such an approach could beconfigured in various ways.

1) A short independent oligonucleotide comprising a perfect complementto the most 3′ residues of the targeting oligonucleotide could beemployed. This oligonucleotide would be sufficiently long that at thereaction temperature it would be likely to form a hybrid with thetargeting oligonucleotide moderately efficiently. This might be between6 and 15 residues in length. The short oligonucleotide will benon-extendable by having a blocking group at its 3′ terminus, such as adideoxy sugar, or other 3′ blocking group. This oligonucleotide also mayrequire a non-phosphate linkage at the ultimate and/or penultimatebackbone link to deter removal of bases in the 3′-5′ direction byediting activities. Although a large proportion of non protein-coatedtargeting oligonucleotides may form duplexes with this shortoligonucleotide this should not significantly decrease the rate andefficiency of the RPA reaction. Firstly, because at the low RPA reactiontemperature there is likely to be an equilibrium between hybridised andunhybridised oligonucleotides there will always be a pool of free meltedtargeting oligonucleotides available. As the oligonucleotide is a bettercompetitor than other random sequences in the reaction, it will befavoured against other transient interactions. Secondly, single-strandedDNA binding proteins such as recA, uvsX, gp32, and E. coli SSB, tend tomelt duplex DNA and thus even if hybrids are relatively stable at thereaction temperature when no proteins are bound, when they bind to thesingle-stranded part of the oligonucleotide and extend cooperatively tothe region of duplex they are likely to enhance its melting, thus theduplex state will tend only to exist on naked oligonucleotides. Finally,recombinases have the capacity to extend strand exchange initiatedbetween a single-stranded DNA region and duplex target into regions inwhich both DNAs are duplex. This appears to be an ATP-dependant branchmigration activity. Taken together these considerations suggest that theshort duplex region should not significantly reduce the rate of the RPAreaction, but instead act to suppress the formation of primer dimer orother artifacts generated from non protein-coated oligonucleotides bybeing a better competitor for binding to the 3′ region of the targetingprimer than other oligonucleotide sequences available in the reaction.If exonuclease deficient polymerases are used, it may be optimal todesign this oligonucleotide to have its 5′ most base pairing to thepenultimate, rather than ultimate, 3′ nucleotide as many polymerasestend to add an additional base to a perfectly blunt end.

2) In a second approach, the targeting oligonucleotide possesses a 5′overhang not present in the initial target DNA, and this overhang is theprecise reverse complement to the sequence to the 3′ end of the sametargeting oligonucleotide, perhaps with the most 3′ base only unpaired(FIG. 35 part D). The length of this complementary oligonucleotideshould be relatively short, but long enough to be a far bettercompetitor than other oligonucleotide sequences present in the reaction.For example between 6 and 10 nucleotides might be optimal. As describedfor the first approach this arrangement is likely to lead to anyuncoated oligonucleotides forming hairpin structures to themselves farmore efficiently than to any other sequences. As the design will placethe 3′ base, or penultimate 3′ base, of the oligonucleotide in a perfectbase-pairing environment, but at the 5′ end of the targetingoligonucleotide, it cannot be extended, apart from the addition of thesingle residue often catalysed by many polymerases if the end is blunt.In this context it may be fine to leave the editing activity ofpolymerases intact. Such hairpin forming oligonucleotides may suppresserroneous activity of naked oligonucleotides without deterring theactivity of protein-loaded filaments.

There are however some important considerations to taking this approach.Firstly, if the recombinase loading is initiated directly on the partlyduplex naked oligonucleotide without initial melting of the duplexsection, recombinase may not extend as close to the 5′ end of thetargeting oligonucleotide as might be optimal. Secondly, as theamplification reaction continues beyond the first round there will beactive displacement of products generated from previous rounds ofsynthesis and if a complete displacement occurs then the very 3′ end ofa displaced strand, which is complementary to immediately adjacentsequences, may be able to hairpin and rapidly self-primer. Such a rapidself-priming event would result in DNA synthesis and formation of anovel double-stranded DNA with both strands joined by a hairpin at oneend. This could be a substrate for further rounds of invasion/synthesisand result in formation of dimer-like products, and possibly morecomplex products (see FIG. 35). We anticipate that this situation may beperfectly acceptable for diagnostic tests. As the amplified sequencesare all dependant upon the presence of bona fide target DNA, and willcontain the unique inter-oligonucleotide sequences, and because theself-priming event may be engineered to function efficiently, then thismay prove an ideal format for diagnostic assays. We have alreadyexperienced the generation of greater than unit length amplified DNAfragment apparently generated from specific targets and suspect thatthis mechanism may operate in the absence of specific oligonucleotidedesign. A similar activity has been described although in this case theactivity was initiated in a totally different manner using a single verylarge single-stranded DNA (Morrical S W, Wong M L, Alberts B M. J BiolChem. 1991 Jul. 25; 266(21):140331-8).

3) In a final approach separate short oligonucleotides with blocked3′-ends would be employed as described in approach 1. In this casehowever a linkage is engineered between the 5′ end of the targetingoligonucleotide and the 5′ or 3′ end of the short competitoroligonucleotide (FIG. 35 parts B and C). This approach is similar toapproach 1, except that by tethering the competitor oligonucleotide inthe close vicinity of the targeting oligonucleotide one ensuresefficient competition of this oligonucleotide with any other sequencesin the reaction.

Polymerase Choice:

There are many DNA polymerases that might be used for RPA. There are,however, a number of criteria that should be considered when designingthe optimal RPA format for a given application. We have identified anumber of different polymerases with activity in RPA reactions, anddeduced which properties confer specific advantages for differentcircumstances. One exciting conclusion is that polymerases fromheterologous systems can be used effectively. We discuss below thepolymerase activities most relevant to RPA.

Polymerase Processivity

Polymerase processivity is measured as the typical number ofincorporation events catalysed on each individual interaction with a DNAtemplate. Many of the polymerase enzymes used in molecular biologyapplications are not highly processive, often because they arefunctional analogues of the of E. coli DNA polymerase I whose primaryrole is DNA repair and Okazaki fragment processing. Processivity is amore critical consideration for RPA than for PCR. Because RPA uses adouble-stranded template, distributive polymerases will producepartially copied strands possessing a joint, which could be ejected bybranch migration. In addition, we have evidence to suggest that bubblemigration may occur in a variety of RPA configurations. In bubblemigration parent strands of the template DNA re-hybridises shortlybehind the replication complex leading to ejection of the newlysynthesised strand, which is freed as a single-stranded DNA instead ofthe outgoing strand of the original recombination event. Thisre-hybridisation has been previously described in the T4recombination/DNA synthesis system and thought to involve coating of theoutgoing strand with the uvsX recombinase and subsequent re-invasion.Alternatively, in the presence of uncooperative single-stranded DNAbinding protein, monomers may be lost progressively from one end of theoutgoing strand and may lead to progressive branch migration running ina 5′-3′ direction, chasing the replication complex.

Thus if the polymerase dissociates prematurely from the template, bubblemigration or branch migration may result in the newly synthesised strandbeing separated from the template as an incomplete single strand. Thiscould be catastrophic for RPA reactions, because if such truncatedproducts are too short to form productive hybrids with similar productsgenerated from the opposing side, these undesired short products willaccumulate linearly. The T4 single-stranded DNA binding protein gp32 canalleviate undesirable branch migration. However RPA urges, wherepossible, the use of relatively processive polymerases, or polymerasecomplexes, to efficiently amplify larger DNA fragments. Several nowcommonly used simple polymerases such as Phi-29 DNA polymerase, Bst DNApolymerase and T7 DNA polymerase in complex with thioredoxin are knownto be processive. However not all of the known classes of relativelyprocessive polymerases may possess the combined additional propertiessuitable for RPA; Phi-29 polymerase has not proven able to supportgeometric RPA amplifications to date, possibly due to an inability toefficiently load onto the recombination intermediates. T7 DNA polymeraselacks sufficient strand displacing activity to be effective. In contrastwe surmised that related Pol I enzymes from the Bacilli, such asBacillus subtilis PolI (Bsu), would likely demonstrate optimalcharacteristics by virtue of sharing with Bst polymerase relativeprocessivity and exonuclease minus status, but retaining optimalsolubility and activity profiles at temperatures in the 30-37° C. range.Additionally, multi-subunit replication complexes incorporating slidingclamps could be used such as that from bacteriophage T4, E. coli, andothers, however assembling all of these components in effective in vitroreactions that maintain the excellent signal:noise and kineticproperties of current RPA reactions remains challenging. We purified thePolymerase I from B. subtilis which is related to Bst polymerase. Thispolymerase is readily overproduced and purified from E. coli with anN-terminal hexhistidine tag, and appears to possess ideal biochemicalattributes for RPA.

Finally, there are some situations where a distributive polymerase maybe more appropriate for RPA. Because in many configurations DNAsynthesis is initiated from opposing ends and replication complexes movetoward one another, there is the possibility of a collision betweencomplexes resulting in a stalemate in which neither progresses further.This requires either that one polymerase temporarily dissociates fromthe template, or that the polymerases used are able to pass one anothereffectively without dissociation. In consequence of the varyingrequirements for an ideal polymerase for RPA we suggest thatexperimental evidence will be the best guide, and to date this impliesthat Pol I class enzymes from eubacteria, in particular from Bacilli,currently show the best properties.

3′-5′ exonuclease activity present associated with the DNA polymerase:

Many DNA polymerases possess 3′-5′ exonuclease activity, and some alsopossess 5′-3′ exonuclease activity, which is probably undesirable in RPAas it results in digestion of one DNA strand progressively as thepolymerase moves forward, rather than displacement. The 3′-5′exonuclease has potential advantages as well as its, obviousdisadvantages. On the one hand 3′-5′ exonuclease activity increases thefidelity of the replication reaction, and can also prevent stalling ofpolymerases at points of misincorporation. High fidelity amplificationis desirable for many DNA applications. The 3′-5′ exonuclease activitymay also be appropriate for amplification of larger DNA fragments wherestalling due to misincorporation could inhibit effective amplification.

Despite these clear advantages of 3′-5′ exonuclease activity there aresome disadvantages. We have observed that the free oligonucleotides canbe subject to end-dependant degradation when polymerases possessing3′-5′ exonuclease are employed. This can be suppressed to a large extentby using saturating amounts of relatively cooperative gp32 protein withsome polymerases such as the Klenow fragment, but with enzymespossessing aggressive exonucleases such as T4 DNA polymerase or Phi-29DNA polymerase, gp32 appears to be insufficient and the oligonucleotidesappear to be completely degraded. These data argue that it isadvantageous to at least limit to some extent the efficacy of anexonuclease activity in the reaction.

We have found that 3′-5′ exonuclease activity of some polymerases maycontribute substantially to noise in the reaction. At the relatively lowtemperatures used in RPA reactions there is a significant tendency foruncoated single-stranded DNA molecules to form inappropriate hybrids, atlow complementarity, with other DNAs in the reaction. Such hybrids willprime DNA polymerases elongation. For extension to occur the last baseor two must be paired correctly with its complement. While poorlycomplementing segments within or between oligonucleotides can form weakhybrids at low temperatures these will rarely be combined with a goodhybrid match at the very 3′ end. Regardless, in the presence of a 3′-5′exonuclease activity the unpaired 3′ most bases will be excised until acorrectly paired 3′ end is formed, as happens normally when an incorrectbase is inserted by the polymerase. Our data suggest that partlyextended strands that have been displaced by branch migration or bubblemigration can fold back onto themselves leaving an unpaired 3′ end,which is trimmed thus promoting inappropriate polymerase elongation.There are therefore good reasons to limit or remove exonucleaseactivities from polymerases used in RPA. There are other methods toinhibit oligonucleotide degradation that may also be used.

Access to 3′ Ends

The recombination intermediate formed after invasion must be accessibleto the DNA polymerase. The structure near the 3′-end of the targetingoligonucleotide, is not equivalent to the 3′-end of an oligonucleotidehybridised to otherwise single-stranded DNA, which is the situation inPCR. Instead, the outgoing strand, which is hybridised to the templatestrand immediately, or shortly, downstream of the 3′-end of the invadingoligonucleotide may block polymerase loading. Moreover, whether of theoutgoing strand is constrained or unconstrained may affect the capacityof certain polymerases to load successfully. Whether a particularpolymerase can function effectively in these situations must beaddressed experimentally. We find that the Klenow fragment of E. coliDNA polymerase I, as well as the Bst DNA polymerase purified fromBacillus stearothermophilus, can load onto and extend such recombinationintermediates. Helicases such as the T4 dda helicase and T4 gp41helicase may also function to process recombination intermediates andseparate the template and outgoing strands downstream of the exchangeevent permitting other polymerases to be used, but these helicases mayalso interfere with other aspects of RPA (see below). Finally, it may bebeneficial to use mixtures of polymerases, acting synergistically in theRPA reaction, for example one polymerase efficient at accessing the 3′ends of recombination intermediates, and the other possessingprocessive, strand-displacing synthetic activity.

Cooperative Component Interactions

We have demonstrated that enzymatic components from heterologous systemscan be combined together effectively in various RPA formats. Forexample, both the Klenow fragment of E. coli DNA polymerase I, the BstDNA polymerase of Bacillus stearothermophilus, and the large fragment ofBacillus subtilis polymerase I, can extend recombination productsgenerated with the uvsX recombinase of bacteriophage T4 in the presenceof the bacteriophage T4 gp32 protein. This suggests that the class ofpolymerases similar to E. coli Pol I are generally effective in RPAreactions, and this may reflect their distinctive properties as stranddisplacing enzymes, combined with access to recombination intermediatesneeded for their repair activities. Furthermore, that elongation ofrecombination intermediates can easily be mediated by polymerases fromwidely different structure groups is uncertain. Despite earlyindications from end-directed recombination intermediates stabilized bybackfire synthesis, we have been unable to drive RPA reactions usingPhi-29 polymerase, and this may reflect that this enzyme cannot readilyload onto recombination intermediates. There may be additionalsynergistic effects when proteins from the same organism are employedtogether. For example there are known to be physical interactionsbetween the bacteriophage T4 components such as uvsY and uvsX, as wellas gp32. This might be extended to other components, foe example the T4polymerase functionally interacts with the gp41 helicase, and physicallywith gp32 (Formosa and Alberts PNAS 80, 2442-2446, 1983). Howeverdespite the seeming attractiveness of using components from the sameorganism to enhance RPA efficiency, we have found that one helicaseemployed for processive DNA synthesis by T4 polymerase may not combineeasily with the effective RPA environment as established here. Earlyexperiments suggested that inclusion of T4 dda helicase may disrupt thecoating of oligonucleotides in the RPA environment and lead to excessiveprimer noise preventing sensitive RPA reactions. This is consistent withan earlier published report [insert reference JBC. 1991 May 25;266(15):9712-8 Inhibition of protein-mediated homologous pairing byb aDNA helicase. Kodadek T.

Resolution of Replication Complex Collisions

In some formats of RPA, such as when a large DNA product is desired, aprocessive polymerase would be the optimal choice. Under theseconditions, however, there is significant likelihood that replicationcomplexes will converge with one another on the same template. There isa danger that replication complexes will become locked head-to head sothat neither can pass. Most useful in this situation are polymerasesthat are both processive and able to resolve collisions as has beendemonstrated for Phi29 DNA polymerase (Elias-Arnanz M, Salas M. EMBO J.1997 Sep. 15; 16(18):5775-83). Alternatively a fine but critical balanceof an ideal level of processivity to permit useful replicative runscombined with a distributive capacity to dissociate fairly frequentlymay solve this issue.

Recombinases

We have assayed both E. coli recA protein, and bacteriophage T4 uvsXprotein in RPA experiments. Both these proteins share some limitedprotein sequence homology and are believed to have evolved from a commonprogenitor. Crystallographic and electron microscope studies ofnucleoprotein filaments of these proteins, which show a conservedfilament structure, in terms of the pitch of the helices formed in bothATP and ADP bound states, suggest a remarkable similarity in theirmechanism of action. Furthermore, all prokaryotes possess proteinshighly homologous to recA, which suggests that the principle activitiesof recombinases have been conserved throughout evolution. Hence, what islearned from one recombinase may be applied to, or substituted by,another.

In addition to their similarities, however, there are differencesbetween recA and uvsX relevant to RPA and as additional components ofrecombination/replication machinery are used in RPA reactionsorganism-specific protein-protein interactions may have a significanteffect on reaction efficiency. The nucleotide hydrolysis rate of uvsX is10-20 times higher than that of recA, suggesting that it might performrecombination reactions at an accelerated rate (Formosa T, Alberts B M.J Biol Chem. 1986 May 5; 261(13):6107-18.). An increased hydrolysis ratecould be beneficial for RPA reactions in several ways.

1) More dynamic turnover of uvsX on oligonucleotides could increase theoverall regeneration of nucleoprotein filaments leading to near completerecombinase coverage to the most 5′ end of invading oligonucleotides.

2) More rapid completion and disassembly of recombinase from successfulrecombination events will permit more efficient polymerase access

3) A more active recombinase will produce a more flexible nucleoproteinfilament.

Another major difference between uvsX and recA is that uvsX hydrolysesATP to ADP plus phosphate, and to AMP plus pyrophosphate whereas recAand other recombinases do not (Formosa T, Alberts B M. J Biol Chem. 1986May 5; 261(13):6107-18.). The biological significance of this differenceis not known but the activity might affect RPA efficiency. For instance,the pitch of nucleoprotein filaments formed with ATP and ADP isdifferent and the hydrolysis of ATP to ADP is associated with overallfilament flexibility. It may be that AMP-bound uvsX can adopt adifferent pitch and possess a distinct flexibility.

The bacteriophage T4 recombinase uvsX stimulates a mode of DNAdisplacement following synthesis known as bubble migration. In bubblemigration uvsX assembles onto the outgoing strand and mediates are-invasion of the outgoing strand thus displacing the newly synthesisedstrand. This mode may have biological significance because by displacingthe newly synthesised strand, the length of region with topologicalconstraint is limited. This process has not been described for recA,although it may occur. Nevertheless several aspects of bubble migrationsuggest real differences between uvsX and recA. For example, the bubblemigration model suggests that it is possible that uvsX bound DNA extendsto the end of the invading or partially extended DNA, but that thisstructure is still accessible by polymerases for elongation (Formosa T,Alberts B M. Cell. 1986 Dec. 5; 47(5):793-806). This has certainly notbeen observed for recA, and if anything there is evidence that may be tothe contrary (Xu L, Marians K J. J Biol Chem. 2002 Apr. 19;277(16):14321-8). It is unclear how similar uvsX and recA filaments arein their respective abilities to promote polymerase loading at 3′ endswith or without recombinase dissociation. We have found differencesbetween uvsX(C) and recA(C) consistent with the notion that theirdifferences have can affect RPA efficiency. Contrary to our findingswith recA, uvsX can mediate multiple invasions to an end structuretarget even when N-terminally tagged gp32 is used in the absence ofpolyethylene glycol (FIG. 23). Thus uvsX may be more optimal for use inRPA.

The bacteriophage T4 uvsX recombinase has a well-characterisedinteraction with its partner loading protein uvsY. Despite reportssuggesting that E. coli recO and recR may be functional analogues ofuvsY we have not observed significant improvement for RPA reactions.This may be due to problems with our protein preparations, or due to theuse of the heterologous gp32 rather than E. coli SSB.

Finally uvsX is likely to behave better with gp32, which we find to bean optimal single-stranded DNA binding protein, because they haveevolved to function in concert and may have relevant interactions.Indeed uvsX and other components of the bacteriophage T4 recombinationdependant replication machinery, such as the dda helicase, have knownprotein-protein interactions that may useful for establishing an optimalRPA reaction (Hacker K J, Alberts B M. J Biol Chem. 1992 Oct. 15;267(29):20674-81).

Despite the apparent advantages of uvsX for RPA, there are features ofE. coli recA that may be useful. It has been reported that recAnucleoprotein filaments are more stable, which could be of utility insome circumstances. The lower ATP hydrolysis rate places less strain onestablishing a durable ATP regeneration system, and the lack ofgeneration of AMP and pyrophosphate obviates the need to regenerate andmop-up these side-products. It may also be the case that other recAhomologues possess activities that are optimal or RPA.

Establishment of a Dynamic Recombination System:

To make RPA robust, it is critical to configure the reaction to providea sufficient number of active, coated, homology-searchingrecombinase/DNA filaments. In addition, following completion ofhomology-searching, filaments must efficiently disassemble, or beotherwise processed, to permit loading DNA polymerase and othercomponents. It is also essential that a sufficient quantity ofsingle-stranded DNA binding protein be present both to facilitateoligonucleotide melting and to collect displaced outgoing strands. Andfinally, robust RPA requires will require processive strand-displacingDNA synthesis. Underlying these requirements is a competition betweentwo the recombinase and the single-stranded DNA binding protein.

It is widely known that of recombinase-loaded DNA filaments are unstablein the presence of single-stranded DNA binding proteins. Coupled to thefinding that nucleotide cofactor hydrolysis is not strictly required forhomology searching, led to the use of non-hydrolysable nucleotideanalogues of nucleotides such as ATP-γ-S, to load recA onto filamentsand produce stable homology-searching complexes. A recA-mediatedamplification method using ATP-γ-S has been described (Zarling, et al.),however, has not been widely used. We previously identified a flaw inthe method, which we can now observe in our experimental results. TheZarling, et al. method probably fails because recombinase-loadedfilaments needs to be dynamic and capable of disassembly as well asother ATP-hydrolysis dependant events to complete strand exchange andpermit loading of DNA polymerase other components to the 3′ end of theinvading oligonucleotide. The use of ATP-γ-S, as well as othermodifications such as removing acidic sequences from the C terminus ofrecombinases, leads to a constant general high affinity for DNA thatlikely prevents strand exchange and dissociation from the invasioncomplex. Thus ATP-γ-S-loaded recA filaments become effectively lockedonto the target site in the recombination event for an abnormally longtime. Consequently non-hydrolysable nucleotide analogues are notgenerally permissive for recombinase-mediated replication andamplification. Instead, ATP, or other hydrolysable nucleotides capableof supporting recombinase loading, must be employed. In ATP therecombinases are constantly associating with and dissociating fromoligonucleotide and are in competition with single-stranded DNA bindingproteins. We have addressed the problem this competition poses in twogeneral ways; first by including a recombinase loading protein specificfor uvsX, the uvsY protein, and secondly by modulating the cooperativebehaviour of gp32, and the recombinases uvsX and recA, by mutationand/or inclusion of crowding agents. It is possible however that limitedquantities of non-hydrolysable analogues such as ATP-γ-S, or ofnon-phosphorylatable analogues such as ADP-β-S, may be included tomodulate the global loading/unloading activity of the recombinase.

Additional Reaction Components

A number of specific reaction components have a significant influence onRPA reaction efficacy.

Polyethylene Glycol

Polyethylene glycols (PEGs) have a profound effect on recombination/DNAsynthesis. Firstly, we find that PEGs influence the number of multipleinvasion/extension cycles that occur, for example when recA is combinedwith gp32(N). We have also found that PEGs stimulate amplificationreactions configured in several different ways (FIG. 15, Example 3). Wealso know that in some configurations PEGs alter the length distributionof products formed (FIG. 28). In summary polyethylene glycols, andpresumably other similar crowding agents may affect the cooperativity ofgp32 and recombinases, affect polymerase processivity and affect thehybridisation rate and behaviour of oligonucleotides in solution. Theymay imbue cell-like environments by phase partitioning the reactants,and/or causing the development of fractal-like kinetics. Furthermore thechain length of the polyethylene glycol appears to be critical. We find,that of those tested, PEGs of average molecular weight 1450 and15-20,000 (PEG ‘compound’) produce the best results. The PEGs in aidinggp32 function, particularly gp32 variants with attenuated cooperativity,has been detailed above. PEG is also likely to increase the stability ofrecombinase-loaded filaments and the increased persistence may increaseRPA efficacy. Most importantly we have been completely unable toestablish amplification conditions capable of amplifying from traceamounts of sample to detectable levels without employing polyethyleneglycols (poorly with PEG 1450, very efficiently with PEG compound).Presumably other agents may also stimulate RPA reactions, however thiseffect was not evident in preliminary experiments with polyvinylalcohol. Thus specific features of polyethylene glycols, and moreparticularly specific variants of PEGs, may be essential.

In what manner PEG compound (carbowax 20M) achieves such a significantstimulatory effect is mysterious. Kinetic models of the manner in whichvolume exclusion may affect reaction rates would place significantemphasis on the molecular weights of the substrates and enzymes understudy in comparison to the crowding component. However the next-mosteffective agent that we tested, PEG 1450, was also the smallest andstimulation by polyethylene glycols intermediate between PEG 1450 andPEG compound were less effective. Consequently we suspect thatproperties additional to simply average chain length are critical to theeffects of these agents. For example specific phase transitions mayoccur at variant temperatures for different PEGs. It is also known thatthe alcohol groups of PEGs can lead to additional interactions, such aswith proteins, or influencing the ionic environment. We note (data notshown) that the addition of 5% PEG compound (while developing a pH below7.0 during storage at room temperature) to Tris-buffered solutions usedin RPA causes a sharp rise in pH.

As a consequence there is currently no formulaic manner to determinewhich volume-excluding agents will have the current properties tostimulate high-level dynamic recombination environments, and RPA inparticular, except through experimental validation.

ATP Regeneration System Components

An ATP regeneration system is crucial to permit persistent recombinationreactions as recombinases have an extremely high rate of ATP hydrolysiswhen bound to nucleic acids. In particular, the uvsX protein has ahydrolysis rate 10-20 times higher than recA and can consume 200molecules of ATP per minute per monomer. A number of systems areavailable and we have routinely used the creatine kinase/phosphocreatinesystem. When uvsX is employed the AMP that is produced must be convertedinto ATP. We have used chicken myokinase, which converts a molecule ofAMP and one of ATP to two molecules of ADP. ADP is then converted to ATPusing the creatine kinase/phosphocreatine system. Poor regeneration ofATP will reduce the reaction rate and likely stop the reaction beforedetectable levels of product have accumulated.

Pyrophosphatase

Pyrophosphate (PPi) accumulates during DNA synthesis and when uvsX isemployed, as it hydryolyses ATP to AMP+PPi. PPi accumulation in thereaction can have several detrimental consequences. Significantpyrophosphate accumulation will permit an unacceptable rate ofpyrophosphorylsis, whereby the synthetic reaction of a polymerase isdriven into reverse and removes the 3′-most nucleotides from duplextemplates. This could lead to unacceptable levels of primer noise, orenhanced levels of undesired self-priming of outgoing strands, becauseediting activities of the polymerase tend to trim 3′-ends back until asuitable duplex region is revealed to permit rapid elongation.Additionally pyrophosphatase accumulation will lead to inhibition of therecombinase, and to a slowing of the polymerase synthetic reaction.

Improved Signal:Noise by Design of RPA Reactions

RPA displays high of sensitivity and specificity. However samplescontaining no target at all often generate products derived only fromthe primers. Such phenomena are not uncommon in other methods. Forexample the PCR method will ultimately generate non-specific productssuch as so-called ‘primer dimers’. RPA is somewhat prone to suchprimer-related artifacts because there is no cycle control allowingsmall amplicons to achieve many doublings within the period of theincubation. Electrophoresis of reaction products permits easy separationof signal and noise, however in simple non-laboratory diagnosticproducts other approaches must be taken. Here we disclose approaches tomake such non-gel diagnostic tests function with adequate signal tonoise, and to easily carry out detection. We also disclose thecomposition of a reaction lyophilizate that can be stored for many daysat ambient temperatures, a prerequisite for easy non-laboratory use.

Signal to Noise Ratios in the RPA System

DNA amplification systems generally contend with the fact thatamplification of DNA may occur which does not initiate from the bonafide target. This ‘noise’, particularly apparent under low targetconditions, may impose restrictions on the permitted sensitivity, andmethods to assess amplification. While initial configurations of RPAoffer exquisite sensitivity we believe novel properties of therecombinase-based system lend themselves to previously untappedapproaches to improve specificity, and to developing novel productdetection schema.

RPA amplification reactions, like PCR, are generally established bycombining two oligonucleotides, which flank the desired target DNA.Doubling events occur and, as in PCR, exponential DNA amplificationensues, permitting as much as 10¹²-fold amplification for fragments of˜150-400 bp.

When primers of 30-35 residues and acceptable to standard designalgorithms are tested, a significant proportion amplify target with highsensitivity and specificity (FIG. 41). For example, such ‘good’ primerscan successfully amplify targets from starting target concentrations of2-3 copies per microliter to generate significant levels of the correctamplicon. However below this copy density even good primers tend togenerate noise (smear, laddering, and/or well staining), as assayed bygel electrophoresis of all reaction products (see FIG. 41), theseproducts apparently of purely primer origin (although contaminating E.coli DNA is likely present and could in theory be specifying highlyinefficient spurious amplicons).

In some cases target DNA may not be particularly rare, for example ifone wishes to determine a limited SNP profile of a human individual anda modest blood sample can easily be obtained. On the other hand, sometests for pathogens may need to detect just a few copies to be of greatpractical utility, and in particular distinguish this situation clearlyfrom complete absence of target.

Our efforts to determine the level and nature of noise generated in verylow, or no, copy number experiments, as well as other experimentsinvestigating primer length and composition, has revealed a number ofkey observations, which we believe will aid the development of idealhighly sensitive portable diagnostic systems using RPA.

Firstly we have found that oligonucleotides of less than approximately30 residues are less effective at RPA, and notably become less ‘noisy’,generating no visible noise at all when reduced to 25-26 (see FIGS. 45and 52). Nevertheless shorter oligonucleotides are still capable of, atleast, hybridization based elongation (see FIG. 52), and their‘activity’ in (recombinase-mediated) RPA events is probably not zero,instead decreasing progressively with shortening (FIG. 45). This permitssome degree of activity/noise tuning.

Secondly, we note that primers which are identical except for additionalsmall number of residues at the 5′ extent show significant variabilityin noisiness (even when all are >30 residues), implying that the exactnature of the 5′-most base(s) plays a significant role in the mechanismand likelihood of noise (see FIGS. 42, 45 and 47).

Thirdly, primers possessing a locked nucleic acid (LNA) sugar at the3′-most end reduce/eliminate noise as determined by gel electrophoresis.They also reduce the quantity of product slightly if only one isemployed, and significantly if both are used at ‘standard’ polymeraseconcentrations, but remain fully active if polymerase concentration isincreased, and under the elevated polymerase concentrations noise stillappears suppressed.

Finally low-target concentration noise appears to derive mostly/solelyfrom primers, not sample DNA, and we thus refer to this noise as primergymnastics. This is clear from experiments in which sample DNA isdecreased to zero. Consequently it is possible to focus attention on thelimited mechanisms by which primers may interact with and betweenthemselves to find solutions to this problem. How noise may arise isdiscussed in detail in the following section. An important conclusion ofthis analysis is that lowering nucleoprotein filamentrecombination/priming activity may impinge more severely on primer noisegeneration than bona fide amplicon generation.

Analysis of how Primer Noise May Originate

Unlike PCR, or other methods involving conventional hybridization, RPA'sreliance on the formation of extended nucleoprotein filaments undergoingATP hydrolysis as active homology-searching complexes likely impinges onthe parameters determining oligonucleotide activity in ways previouslyunencountered for in vitro amplification reactions. We suggest that therapid drop in oligonucleotide activity below 30 residues described abovereflects recombination rate slowing, and that most likely this parallelsobservations made elsewhere on the length-dependance of ATP hydrolysisfor the E. coli recA recombinase (Bianco P R and Weinstock G M). Inparticular the length and composition of short sequences was shown toinfluence ATP hydrolysis. Roughly 30 residues or more are required formaximal ATP hydrolysis (moles ATP per mole DNA-bound recA) when recA isbound to synthetic oligonucleotides, shorter sequences displaying markeddrops in hydrolysis rate until it was absent by about 15 residues. Weconnect the observation of low amplification behaviour with previousdata on ATP hydrolysis because there is a clear rationale to think thatnucleoprotein recombination activity will be influenced by ATPhydrolysis rate. In a sense the slowest oligonucleotides we haveobserved are those bound with unhydrolyzable ATP-γ-S in which the rateof completed exchange (formation of resolved plectonemic joints) is nearzero (Riddles P W and Lehman I R) We have also proven that ATP-γ-Seffectively poisons RPA reactions (Piepenburg et al.). We suggest thathigh rates of ATP hydrolysis in a nucleoprotein filament underpins theactivity of ‘fast’ primers, and low rates ‘slow’ primers, who differprincipally by the average number of complete recombination eventscompleted in a unit time. Interpreted simply, ATP-hydrolysis is requiredfor dynamic activity of filaments. Oligonucleotides of less than 30residues become more stable and undynamic, eventually resembling thelocked state generated when ATP-γ-S is employed, and are unable tocomplete exchange (‘slow’ oligonucleotides may locate sequences asreadily but be unable to disassemble readily to permit completion andpolymerase access).

A Possible Difference in Dependence on Nucleoprotein Filament Activityfor Boa Fide Product and Noise

To understand why there might be any advantage to selecting less active‘slower’ oligonucleotides we need to explore how primer ‘gymnastic’noise might arise. Why might shortened oligonucleotide reduce noisefaster than the specific signal? Unlike the geometric phase ofamplification seen for both bona fide targets and noise products, theearly formation phase of noisy targets from starting primers is probablymore complex, and is presumably rather slow. In FIG. 43 we have detailed2 general mechanisms by which individual primers can become converted tostructures with the capacity for geometric amplification. Bothstrategies diagrammed illustrate the formation of amplicons from asingle primer species. In both cases the first step involvesself-priming of the oligonucleotide by formation of a short hairpin atthe 3′-end. In strategy 1 the resulting hairpin will not easily amplifyin geometric phase because of significant mismatches. Instead to becomegeometric phase amplicons they would need to proceed through a step moreakin to that described in strategy 2. In this case the firstself-priming event is followed by a second involving the reversecomplement to the 5′ end of the original oligonucleotide. There areseveral simple remarks from such modeling. First is that 2 types ofevent have to occur; there must be elongation from the 3′-end of anoligonucleotide, and then a second similar 3′ hairpin and extensionwhich involves the reverse complement to the 5′-end of the originaloligo. Note that the very rare and unstable nature of the first eventspreceding geometric phase are going to be most strongly inhibited ifmost oligos are coated with either an SSB or recombinase, which have DNAmelting activites when bound to ssDNAs. Conversely dynamic recombinasefilaments which disassemble spontaneously create time windows in whichall, or parts, of an oligonucleotide are uncoated. This very ratelimiting step will therefore likely be sensitive to primer ATPhydrolysis rate, dynamicity, and by extrapolation oligonucleotidelength. Consequently shortening oligonucleotides may be particularlyeffective and stemming noise, sufficiently so to justify slowing bonafide target amplification rate in order to achieve it.

These final geometric phase amplicons are inverted repeats. This meansthat an invasion/elongation originating from one side will displace astrand that immediately folds back on itself to form a long hairpin.This molecule must be targeted by recombinase action in order to beconverted back to one similar to the parent, while bona fide targets canemploy solution hybridization additionally. This imposes additionalnecessity for the primer artifacts on recombinase-based invasion toachieve geometric amplification, and consequently they will probably besuppressed more rapidly by lower recombination rate. Further thepresence of internal repeats means that even larger and more diverseversions of these amplicons are likely to arise quickly, and this isconsistent with the common phenotype of a smear, or ladder, running mostof the way up a gel.

A third remark is that the time taken to double a given species of DNAduring exponential amplification will be a compound of recombinationrate and time taken to synthesise down the template by polymerase. Asprimer noise products (at least initially) are very small, the timetaken for DNA synthesis is very low and so the principle rate limitationmay be the recombination rate. Thus decreasing nucleoproteinrecombination rate will, while slowing amplification in general, impingemore heavily on short amplicons than long ones, thus generallybenefiting bona fide amplicons.

We draw on the above observations to devise ways to combine primers ofdifferent length, composition and concentration, to give highlysensitive and specific reactions.

Primary Oligonucleotide Design:

The four observations described above lend themselves to somestraightforward oligo design principals.

Oligonucleotide Length

Optimal primer length may be around 30 residues and in some cases a fewresidues shorter will yield an excellent signal-to-noise ratio. As theaverage primer length is decreased, the noise generation decreases andseems absent altogether when 26-28 mers are employed (FIG. 45). Althoughthe amplification of the target also decreases, we suspect that noisedecreases more quickly than signal. In the previous analysis of hownoise may arise, we conclude there are several reasons why primer noisemight be more affected by decreased recombination rate than correctamplicons. 5′-Most Sequence Optimization

By comparing the activity of oligonucleotides possessing identicalsequences except for the extent of the 5′-most residues we have notedthat significant variation in oligonucleotide noisiness is observed (seeFIGS. 42, 45, 47). One possibility is that this purely reflects theeffects of length variation on nucleoprotein activity as describedabove. This may not be, however, the only reason if primers beingcompared are all 30 residues or longer. For example in FIG. 42, theoligonucleotides NEST-1 and J1 are identical except that J1 containsseveral extra 5′ residues. Instead of being noisier, thisoligonucleotide is less noisy, while amplifying very well. Based on thisand other cases, we propose two other likely mechanisms. First, that anoligonucleotide to become exponentially amplifiable may require apriming event by the reverse complement of the 5′-most sequences, thisevent critically depends on the exact sequence of bases at the 5′-end.This means that varying the 5′-most bases may lead to significantvariation in noise generation. Second, that the base composition of even5′ sequences, even when the oligonucleotide is great than 30 bases, mayaffect the recombination activity of primers. Evidence to support thiscomes from an experiment in which 5′tags consisting of homopolymericstretches were added to primers, and the consequences observed. FIG. 47shows an experiment in which a run of C residues, or G residues, wereappended to the oligonucleotides J1 and K2, and subsequentlyamplification reaction were established containing all possible modifiedand unmodified oligonucleotides alone, or in the possible combinations.Surprisingly these oligonucleotides, already determined to be verysensitive and specific, were not improved by adding these particularhomopolymeric stretches. Instead it was observed that if a run ofCytosines was added to the 5′ end this lead to increased production ofnon-specific products when incubated alone, while a stretch ofGuanosines had the reverse effect, making the oligonucleotides veryquiet when left alone. Also, apart from the combination of the originalparent unmodified oligonucleotides, the only pair to apparentlysuccessfully generate a correct product was the combination of the 2Cytosine-tailed oligonucleotides (i.e. the most noisy oligonucleotides).One explanation that can justify these observations is that a stretch ofCytosines at the 5′ end makes the consequent nucleoprotein filament moreactive, while Guanosines make it less so. In summary oligonucleotidecomposition, like length, may affect rate behavior, and additional andunrelated sequences at the 5′ end can have a significant influence inthis respect. Optimal primers may be found by a rational approach to 5′sequence design. Several different 5′-most bases might be tried, and thebest selected for employment. Additionally it is likely that tags can beplaced at the 5′ end whose sequence is unrelated to the target, butconfer resistance to snapback hairpin priming from the reversecomplement. Determination of optimal sequences that work on manyoligonucleotides may be possible. Measurement of ATP hydrolysis rate ofoligonucleotides in the presence of reaction components may also proveof utility in designing optimal primer pairs, and permit one to createprimer mixes which all share identical amplification rate behavior. Likelength, these observations and improvements would not be obvious inother systems because they depend on specific features of arecombinase-driven amplification system and the biochemistry thatunderlies it.

Locked Nucleic Acid, Ribose, or Other Sugar Modifications

Locked nucleic acids (LNAs) are nucleotides where a linkage has beenengineered between the 2′ and 4′ Carbons of the sugar ring of ribose.Oligonucleotides containing such sugars, most notably at the very 3′position, have been successfully used in PCR assays, and shown tosignificantly improve the discrimination of correct 3′ base pairing (seeVester B. and Wengel J.). We anticipated that LNA residues might betolerated by the RPA system providing that they were not present at toomany position within the oligonucleotides. As they are known to increasespecificity in PCR amplifications they might well improve signal-noisein the RPA system.

Earlier experiments employing phosphorothiorate linkages at the most 3′positions proved unable to function properly in RPA, and were notablymore noisy (data not shown). We interpreted this as a reflection thatboth uvsX and gp32, which interact with the sugar-phosphate backbone,would not bind these backbones leading to seriously anomalous reactionbehavior. However gp32 binding is less dependant on the sugar asevidenced by its ability to bind to RNA as well as DNA (albeit withaltered less cooperativity) (Kowalczykowski S C, et al.). Thus weanticipated that LNA sugars, which retain a normal phosphate group mightremain functional with gp32.

We have performed experiments in which oligonucleotides possessing alocked nucleic acid sugar at the most 3′ position have been used inamplification reactions. Comparison has been made between reactions withidentical oligonucleotides with and without the sugar modification, andreactions involving one modified and one unmodified oligonucleotide.Based on experiment it appears RPA can amplify DNA when one or botholigonucleotides possess a locked nucleic acid residue at the 3′position. Notably the presence of a LNA nucleotide at the 3′ endsignificantly reduced product accumulation under ‘standard’ polymeraseconcentration, and apparently eliminated associated noise. Very lownoise, and reduced product, may arise because of lower successfulrecombination frequency, reduced polymerase takeoff, or a combination.Whatever the reason it is reasonable based on the findings that usinglocked nucleic acids in one or both primers will significantly reduce oreliminate noise, while retaining an acceptable amplification rate forbona fide product. We found that product levels can be returned to highlevels if the polymerase concentration is increased several-fold (fromabout 30-40 ng/μl to 150 ng/μl), but that noise levels do not rise at anequivalent rate.

Similarly other sugar-modified residues may display improved signal tonoise behaviour when present at some frequency in oligonucleotides. Forexample ribose, 2′-O-methyl modifications, acyclic sugars, or otherwisemay prove of utility.

Approaches that Improve Signal to Noise by Combining NucleoproteinFilaments with Differential Activities

The ability to control the rate of recombination and/or elongation fromprimers on the basis of their length, base composition and distribution,and use of modified sugars (or possibly other backbone modifications)suggests mechanisms to design amplification reactions with optimalsignal to noise properties. There are two general ways in which weenvision oligonucleotide combination strategies to be employed tobenefit. First are strategies in which single tube nesting occurs, andsecond are the employment of a non-noisy oligonucleotide paired with amore conventional oligonucleotide in such a way that bona fide ampliconsare readily formed, but noisy amplicons involve only singleoligonucleotides.

Single Tube Nesting

Nesting is the process by which improved sensitivity and signal to noisehas been enabled by carrying out a first amplification with an outerpair of primers, and then perform a second amplification with an innerpair of primers. In its most familiar context this approach has beenemployed in conjunction with the PCR method. In principal the firstround amplifies the target more efficiently than random DNA, such thatafter the first amplification even if it was insufficiently clean togive a clear amplicon against background, it still is so relativelyenriched that a second amplification with primers internal to for thefirst will readily give a very clear and clean result. In practice thisnormally relies on performing one amplification, then removing a smallportion to a new tube, and performing a second amplification withinternal ‘nested’ primers.

While the concept of nesting is visited elsewhere our results suggest anattractive alternative nesting approach in which all the participatingprimers are combined in a single reaction. The rationale being that theinner and outer oligonucleotide pair are differentially active inrecombination and/or elongation rate, and be present at differentconcentration. For example the outer pair would be configured to have afast recombination/elongation rate, and the inner pair a slowerrecombination/elongation rate. Also the outer pair would be present atlower concentration (but still sufficiently high concentration foracceptable activity) and the inner pair would be present at highconcentration. What would then happen? In isolation the inner pair wouldamplify cleanly but rather slowly so that they might not achieve anacceptable degree of amplification within a desirable timeframe. Incontrast, in isolation the outer pair would amplify quickly, albeit withsome primer noise, but would stop at a rather low concentration due toexhaustion. In combination however these two behaviours wouldpotentially complement nicely. In practice outer primer pairs wouldgenerally be fast, over 30 residues in length, unmodified at the 3′-end,and possibly containing additional 5′ residues that promote ‘fastness’.Inner primers would be slower, as potentially conferred by making themshort, modifying the 3′ end with LNA or otherwise, adding slownesssequences at the 5′ end, and so on.

Non-Noisy/Noisy Nucleoprotein Pairs

Experimental data that we have obtained suggests that much of theprimer-derived smears or ladders contain sequences from one primerspecies only (see FIG. 46). If true this fact alone suggests a simpleapproach to distinguishing signal from noise merely by determiningwhether or not the two primers used in the reaction become physicallyassociated in products. Here we suggest that this can be furtherimproved by combining very quiet oligonucleotides with faster (noisier)oligonucleotides. Quiet oligonucleotides, for example shortoligonucleotide, may still function efficiently if present with noisierones providing that they can function in hybridization reactions (seeFIG. 52). However if they only engage in amplification via normalhybridization whose kinetics are very different to recombinase-promotednoise amplification they may remain uninvolved in the noise that thefaster oligonucleotide engages in alone.

Third Primer and Detection Protocols

The possibility that primers might principally indulge in gymnasticsalone, as mentioned above, suggests simple approaches to determiningwhether or not amplification has occurred efficiently. One simplenon-gel detection format would employ one oligonucleotide labeled with adye or enzyme, or other easily detectable group, and the other labeledwith an immobilizable group e.g. biotin (see FIG. 50 Part 1). Thusbefore, during, or after the main reaction phase the immobilizableoligonucleotide would be immobilized to a surface, and at reaction endthis surface would be washed with an appropriate buffer. If the otherlabeled primer co-associates with the immobilizable primer, which can beeasily determined, then amplification of the product has occurred.

Beyond this we imagine additional ways in which additional levels ofstringency on bona fide product amplification might be generated. Asimple additional approach involves a ‘third’ primer which functions tocapture onto a surface liquid phase amplicons. This primer would bedeterred from generating noise itself either by being engineered asquiet (e.g. a short oligo as described above), or by being added to thesystem only at a late phase of the reaction. This third primer mighttarget novel internal sequences, but also could immobilize the targetsvia ‘backfire’ synthesis described elsewhere to avoid disruptive branchmigration (FIG. 50, Parts 2 and 3).

Lyophilization of the RPA Reaction

In order to configure non-laboratory, or near-patient, diagnostic andforensic tests it will be necessary to provide ambient-stable reagents.One obvious way to achieve this is by lyophilizing the reactions,omitting only the sample DNA, and possibly any other component which isstable separately and can be added with the sample such as buffer usedto dissolve the sample nucleic acid.

While lyophilization is a well-established process there is no guaranteethat all components of a reaction system will successfully byco-lyophilized and reconstituted under the same conditions. We haveattempted to lyophilize RPA reactions with and without various of thefinal reaction components. FIG. 53 shows that we have successfullymanaged to lyophilize RPA reactions containing everything except thesample DNA and some buffer component (which can be stored stably atambient temperatures and used to reconstitute sample DNA). Thedisaccharide sugar trehalose proves in these experiments to be requiredto stabilize the lyophilizate, permitting room temperature storage forat least 10 days.

Real-Time Kinetic Analysis of RPA Reactions

The ability to perform kinetic analysis of DNA amplification reactionsprovides enormous utility compared with purely end-point analysis ofsimilar reactions. For one thing this allows the determination of thenumber of DNA, or RNA, copies present in a sample and has many uses inresearch, clinical, and environmental testing applications. In additionto straightforward quantification applications, dynamic reaction productdetection offer excellent solutions to other problems, such aspermitting the discrimination of single nucleotide polymorphic allelesby virtue of altered accumulation kinetics, and a mechanism to assesspresence versus absence of targets in a non gel-based format.

RPA offers an excellent alternative to PCR to amplify specific DNAtargets, but by obviating thermal cycling, RPA requires less expensiveinstrumentation and is easier to implement in non-laboratory settings.

Currently real-time analysis of DNA accumulation is most widely employedin combination with the PCR. PCR shows exponential amplification of DNAup to several cycles after reaching a product detection threshold andcan be implemented using relatively inexpensive optics when variousfluorescence-based sensors are used. Consequently assessing the cyclenumber a given sample crosses the detectability threshold, incombination with equivalent results from control samples, permitsassignment of the number of copies present in anonymous samples. Anumber of sensor approaches have been described, and many are currentlyemployed. Fluorescent minor groove binding dyes can be employed whichdevelop strong fluorescence once bound to DNA. Examples of such dyesinclude SYBR gold and SYBR green (Wittwer et al. [1]). Additional otherapproaches have been successfully implemented, and these gain fromadding target specificity to the sensing permitting greater sensitivityand specificity, particularly at low target levels in the initialsample. One simple format comprises two oligonucleotide primersrecognising adjacent internal sequences of the amplicon, each labelledwith a fluorophore. If the target is present the two fluorophores becomeclosely situated and fluorescence resonance energy transfer (FRET)occurs, which can be detected with suitable excitation and emissionfilters (Wittwer et al. 1997 [2]). Other preferred systems employ probescombining the presence of both a fluorophore and a quencher. For somemethods hybridisation to the target amplicon causes separation ofhairpin-associated fluorophore and quencher and hence an increase influorescence. In another method hybridisation of the probe is assessedvia the action of an oncoming polymerase possessing a 5′-3′ exonucleasewhich attacks the probe and leads to permanent separation of thefluorophore and quencher (so called ‘Taqman’ probes) [Heid C A, StevensJ, Livak K J, Williams P M., Real time quantitative PCR. Genome Res.(1996) 6:986-94]. Implementation of these approaches and others iswidely documented.

During PCR reactions several distinct and temporally independent phasescan be identified. All DNA is single-stranded at the high temperature(e.g. 94° C.) used to melt all DNA strands. Subsequently amplificationprimers may be annealed at a second low temperature, such as 50-65° C.Finally elongation of primers to generate principally double-strandedproducts is performed at, typically, 70-75° C. Due to the controlledcyclic nature of PCR it is desired and necessary to assess theaccumulated level of DNA at specific distinct points in the cycle. Thismay occur at the end of the synthesis stage. Alternatively it may bedesirable to measure fluorescence at some other point, and at another(fourth) temperature depending on the approach employed.

Unlike PCR, RPA reactions are principally configured to operate at asingle temperature. Current configurations of RPA are unphased andtherefore a complete variety of reaction ‘stages’ will be simultaneouslypresent in a sample (phasing may be attained in theory by approaches,for example, such as controlled uncaging of ATP). As a consequence ofthis lack of phasing at any given moment there is likely to be a mixtureof double-stranded DNA, single-stranded DNA (such as displaced strands,as well as oligonucleotides), and intermediates of heterogeneous nature(such as triplex intermediates and/or homology-searching complexes).Despite the heterogeneity of the reaction mixture it will generally bethe case that the steady state levels of double-stranded DNA, andpossibly single-stranded DNA, will increase during the reaction asproduct accumulates. On this basis it seemed reasonable thatstraightforward approaches to measuring product accumulation might bereadily employed.

Here we describe quantities of SYBR green and SYBR gold dyes that arecompatible with RPA and permit real-time detection of accumulatingreaction products. It is also the case that sequence-specific monitoringprotocols will be compatible with RPA. Such approaches would include theemployment of two fluorescently labelled oligonucleotides, which onhybridisation undergo fluorescence resonance energy transfer, or the useof dual-labeled probes such as those that are quenched untilhybridisation results in alterations in FRET, or they are separated byassociated nuclease activity. We provide evidence that the ‘Taqman’approach which relies on the so-called 5′-3′ exonuclease activity ofcertain polymerases cannot be used in RPA reactions, as these nucleasesare actually structure-specific FLAP endonucleases which inhibit RPAreactions. Furthermore certain approaches, such as molecular beacons, inwhich natural hairpin-forming properties of the probe are essential, maybe less successful in RPA due to likelihood of the probe to be in amelted state in the present of single-stranded DNA binding proteins andrecombinases.

FIG. 54 shows the results of experiments to determine whether SYBR goldand SYBR green stains are compatible with RPA. An initial experimentwith SYBR gold in which various dilutions of the SYBR gold dye were madeform the supplied stock (described as a 10,000.times. stock frommolecular probes in DMSO) was performed using primers to amplify afragment of the human apoliporotein B locus (FIG. 1 A,B). The reactionis clearly inhibited if the final concentration was 1.times. (1:10,000from stock) or 0.4.times. (1:25,000 from stock), but was notsignificantly observed at 0.2.times. (1:50,000 from stock). Thisconcentration of 0.2.times. was then employed as the highestconcentration in a dilution series in 50 microliter reactionsestablished with the same two human-specific primers, at two (low)target concentrations of total human genomic DNA (2 or 20 copies permicroliters starting copy density) (FIG. 54 C). A master mix wasassembled on ice and aliquoted into wells in a 96-well microplate cooledto ice temperature. Once mixed the plate we transferred to afluorescence microplate reader with a stage set to 37° C. The reader wasset to collect fluorescence readings at excitation 485 nm, emission at528 nm, at 1 minute intervals over a period of 1 hour. In the same runSYBR green dye was diluted to the same degree (D) and concurrentlyassayed in similar samples. This experiment revealed several key factsrelevant for configuring real-time RPA reactions with SYBR gold or SYBRgreen. First, direct comparison of detection times within the SYBR goldexperiment shows that apparently even 1:50,000 fold dilutions slow thereaction relative to higher dilutions. Indeed 1:100,000 or 1:80,000 gavesignificantly faster reaction kinetics, and thus would appear to be abetter quantity of SYBR gold to use than the 1:50,000, which appearedacceptable by earlier end-point analysis. However, the relative maximalfluorescence signals obtained with the higher dilutions weresignificantly less than with the lower, suggesting that the dye werebecoming limiting. Also, even at the highest dilution of SYBR goldfluorescence increases over background was detected later than thesamples in the SYBR green experiment, and the overall fluorescencesignals for all SYBR gold concentrations were much less than for SYBRgreen. In contrast the SYBR green samples were in all cases detectedearlier than the SYBR gold experiments. In conclusion we suggest thatwhile both dyes can be implemented in real-time RPA, SYBR green appearsthe more robust for standard analyses. It appears to work well at finaldilutions of between 1:50,000 and 1:100,000, but the more concentratedsamples (1:50,000) gave higher overall fluorescence and longerdetectable exponential phase suggesting that 1:50,000 dilution was thebest in these experiments. We have also assayed with higher SYBR greenconcentrations successfully, but there is some indication thatinhibition may eventually occur. We suggest that between 1:50,000 and1:25,000 is the optimal SYBR green concentration for dynamic real-timeRPA assays.

FIG. 55 shows an example of two experiments to determine the capacity ofthe system to distinguish between different numbers of copies of a DNAtarget (present in B. subtilis genomic DNA). Starting number of templatemolecules of 500,000, 50,000, 5,000,500, 50 or zero copies of B.subtilis DNA were present in the various samples and amplification wasperformed with B. subtilis SpoOB locus-specific primers BsJ1 and BsK2.In many independent experiments the system successfully generatedprofiles similar to the representative experiment shown in FIG. 56. Notethat the spacing between the exponential phase curves is similar between10-fold copy number dilutions as required for a quantitative system. Weconclude that RPA is quantitative over at least 4 orders of magnitude ofstarting template number, but quantitative results over a larger rangemay be possible. Note that in (FIG. 55C), the second B. subtilistitration experiment, the highest target concentration trailed offunexpectedly early. Later experiments indicate that this may arisesporadically as a consequence of the relatively low levels of gp32 anduvsX employed in these experiments compared to later experiments withroughly double the levels (FIG. 56).

We also investigated similarly the ability of the SYBR green RPA systemto monitor a range of starting template copies of human DNA byamplifying with a number of dilutions of human DNA. FIG. 57 shows theresults of such analyses. As expected the data fit the expected profileindicating that RPA behaves in a quantitative manner and can easily beassayed with SYBR green. A similar trailing effect was observed for thehighest target samples, but was corrected in later experiments in whichhigher amounts of gp32 and uvsX were employed (see FIG. 57 C versus B,and the concomitant increase in overall end-point product levels).

We show that using fluorescent dyes it is possible to monitor thekinetics of RPA reactions, and that this in turn can be used to indicatethe quantity of specific targets in a sample. The simplestinterpretation is that double-stranded DNA accumulates in an exponentialfashion in RPA reactions until a mass point above the detection limitsof SYBR green fluorescence. It is possible, however, that thefluorescence profile of SYBR green RPA reactions reflects additionalactivities. For example RPA products may under some circumstances engagein recombination reactions with other products leading to complexintermediates whose SYBR green binding behaviour is not well understood.Also the kinetic profile of product accumulation may alter as aconsequence of reaction ageing phenomena only partly related to thestarting copy number. This may underlie the spurious early fluorescencetrailing observed for the highest copy number samples in some of theseearly experiments, although later experiments with increased uvsX andgp32 levels avoided this phenomenon.

We observed in a number of cases that the water control appeared tobegin rising in a similar timeframe to the lowest concentration ofsample (typically 1 copy per microliter start density, or less), howeverthe kinetics of the water control product accumulation seemed distinctfrom the target-containing samples, having a shallower exponential phaseindicative of slower doubling times. This observation is probably due tostructural properties the amplicons possess (e.g. internal repeats) thatreduce the rate of synthesis in the RPA system. We commented earlier onthe possibility that primer-derived amplicons would tend to containinverted repeat structures that would often require 2 rounds ofinvasion/synthesis per duplex doubling, rather than the single one thatcould suffice for bona fide target amplicons. Regardless of the originof this phenomenon we speculate that with sophisticated data analysissoftware and suitable experimental internal controls, this noise mightbe identified and distinguished from the kinetic behaviour of truetarget amplification. Furthermore we observed some distinct variation inthe time taken to reach detection, and the slopes of the curves, betweendistinct amplicons. This would be expected as variation in the activityof different primers, and the variant lengths and sequence compositionof different amplicons, suggests that average doubling times would varybetween amplicons. The slopes of the fluorescence profiles by thiscriteria alone are likely to vary between different types of amplicons,and this may be usefully employed in analysis.

The experiments performed here suggest that kinetic RPA reactions can beused to decrease the time taken to assess the presence of target DNA insamples compared to later gel electrophoresis. The experimental set-upwe have used here is far from ideal as the 96-well plates used are madeof thick plastic, and the heated plate on the fluorometer is not indirect contact with the sample wells. We estimate that it takes up to 5minutes for a 50 μl volume reaction to attain temperature under theseconditions, and perhaps 8 minutes for a 100 μl volume. In an optimiseddevice these long lag times would not exist. Consequently we estimatethat for a clinically relevant quantity of human DNA (say 1000-3000copies, ˜3 ng-9 ng) amplification could be assessed readily in roughly30 minutes with the appropriate equipment using typical conditions shownhere. Despite the limitations of these experiments using 96-well platesand a conventional fluorometer, these pilot experiments are highlyencouraging and indicate that kinetic monitoring of RPA reactions offersa tractable approach to quantification, and that this is quantificationmay be practically implemented to assess DNA levels over at least 5orders of magnitude.

RPA Reaction Control Mediated by ATP Concentration

RPA is a versatile method, but it can be improved by incorporation offeatures to control precisely when in the reaction recombinases areactive. Such control can be gained through the periodic release of ATPby photolysis of caged-ATP. Alternatively, the reaction concentrationATP or other nucleoside triphosphates, can be cyclically regulated byrepeated manual addition or the use of a biochemical oscillator.

Caged ATP cannot supporting either the DNA binding and recombinasefunction of E. coli recA protein [Butler B C, Hanchett R H, Rafailov H,MacDonald G (2002) Investigating Structural Changes Induced ByNucleotide Binding to RecA Using Difference FTIR. Biophys J 82(4):2198-2210]. Following photolysis, however, released ATP enablesrecombinase function. All prokaryotic recombinases so far studied aredirect homologues of the recA protein with primary sequence homology,and structural homology. Moreover, all recA homologues studied,including the eukaryotic homologues, require binding to ATP to enablerecombinase function. We expect that similar phenomena with therefore bemediated with T4 uvsX and other recombinases accordingly.

The role of nucleotides in regulating recombinase action is relativelywell documented. In the case of prokaryotic recombinases, e.g. E. colirecA and T4 phage uvsX, the recombinases hydrolyse ATP to ADP (and AMPin the case of uvsX). Hydrolysis is occurring with high activity as longas the recombinases are bound to DNA. The ADP-bound state has loweraffinity for DNA and is generally associated with filament disassemblyfrom DNA (and altered nucleoprotein filament pitch). Under typical invitro conditions ATP is maintained at a relatively high concentration inexcess to ADP, to ensure that rapid of exchange of ADP for ATP innucleoprotein filaments deters premature disassembly. Consequently, therecombinases in a reaction respond to the ATP:ADP ratio and to someapproximation net disassembly of the nucleoprotein filament occurs whenthe ADP concentration exceeds the ATP concentration.

RPA reactions rely on the action of recombinases to load onto syntheticsingle-stranded oligonucleotides and carry out homology-searchingactivity. As described the activity of the recombinases is dependant onthe presence of nucleotide triphosphates, most obviously ATP. Whenrecombinases are DNA bound they hydrolyse the nucleotide triphosphate ata high rate, for example the T4 uvsX protein is known to hydrolyse 200molecules of ATP per protein molecule per minute at 37° C. onsingle-stranded DNA (although rates on shorter oligonucleotides may bemore variable). Consequently there is a need for a large supply of ATPto maintain an active recombination reaction in which a large proportionof oligonucleotides are associated with recombinase filaments,particularly when the oligonucleotides are present at near micromolarconcentrations.

Consider the flux of reaction nucleotide levels that might occur duringa typical RPA reaction. If a pair of oligonucleotides is employed in areaction at a concentration of 1 μM each, the oligonucleotides being 35residues in length, then at 10% saturation of the oligonucleotide withrecombinase, there is roughly a bound-recombinase concentration of 2.8μM (a value of 10% saturation in 3-5% PEG compound is roughly consistentwith the results of experiments in our hands, but could be slightlyhigher or lower). This would convert a 0.56 mM solution of ATP to theequivalent amount of ADP every minute based on the published hydrolysisrate. Consequently if a 3 mM solution of ATP was used to initiate thereaction, and no ATP regeneration system were present, then after only 3minutes the ADP concentration would rise to levels equal to the ATP andthe recombinase would become inactive.

We have routinely used a total oligonucleotide concentration of 0.6 μM,and a uvsX concentration of roughly 3 μM. Should all of this uvsX bebound to oligonucleotides it would consume roughly a 0.6 mM solution ofATP in 3 minutes, given a reaction rate 200 molar quantities per minuteon oligonucleotides. Based on our data limited hydrolysis data, however,it is unlikely that complete binding of uvsX molecules tosingle-stranded DNA is ever achieved under our typical RPA conditions.Although it is formally possible that the per monomer hydrolysis rate islower for the length of oligonucleotides we employ. These alternativesare suggested by the fact that in some cases we have been able toamplify a target DNA to levels detectable by ethidium bromide staininglevels without the presence of a regeneration system. Other experimentssuggest that is takes roughly 30 minutes to achieve this level ofamplification, and by deduction we calculate that a maximum of only 0.15mM ATP could possibly have been consumed in each 3 minute period, aquarter of the predicted level).

Analysis of effective concentrations of uvsY in reactions is consistentwith the expected stoichiometries. We have routinely used roughly a halfto equi-molar concentrations of uvsY compared to uvsX protein, andpublished data suggests that uvsY protein probably functions as ahexamer. If this is so and one hexamer is required to load, andstabilise, each loaded filament, then whereas there would be a need forroughly 12-14 uvsX molecules per oligonucleotide (30-35 mer) there wouldneed to be 6 uvsY molecules per oligonucleotide, i.e. roughly half themolar concentration. This experimentally determined optimum satisfiesnicely the theoretical prediction that half to equi-molar concentrationsof uvsY to uvsX are required.

In summary, the conversion of ATP to ADP in a typical uvsX-supported RPAreaction with 0.6 μM oligonucleotides, 3 μM uvsX, and 1-3 μM uvsY isabout 50 μM per minute and no more than that. Roughly 16 molecules ofATP are hydrolysed per uvsX molecule per minutes in the reaction. Thisfigure is six-fold less than that predicted if all uvsX molecules werebound to ssDNA and hydrolysing ATP at 200 molecules per uvsX per minute,and presumably reflects either that only a fraction of the uvsX is boundat any one time, that hydrolysis rates are lower on shortoligonucleotides, and/or that in reactions with no regeneration thehydrolysis rate falls off significantly as the ADP levels rise. Wesuggest that while the last factor may become important eventually,during most of the reaction this is not the primary factor.

Having deduced experimentally the consumption rate of ATP in a typicalRPA reaction, it now remains to estimate what size pulse of ATPconcentration we would need to use to stimulate suitable bursts ofrecombinase activity. To do this we need some estimate of the K_(m) fora particular recombinase.

The K_(m) of ATP hydrolysis by recA is reported to be 20 μM.Consequently relatively little free ATP needs to be released into arecombinase system to promote activity. Assuming this is also true foruvsX, a pulse of ATP that changes the reaction concentration from zeroto 50 μM would amply support homology searching until such time that theADP level accumulates to a 1:1 ratio. Under standard conditions thiswould be roughly 30 seconds and would eventually produce 25 μm ADP and25 μM ATP. A 30 second burst of recombinase activity is likely to belonger than what is required for a round of invasions to occur.Additional pulses of ATP can readily generate additional bursts ofrecombinase activity. For example a further pulse of 50 μM rise wouldraise the ATP level to 75 μm compared to 25 μM ADP. After 30 seconds theATP level will equilibrate to 50 μM ATP and 50 μm ADP and the reactionwill again halt. A further pulse will raise the ATP to 100 μM and theADP to 50 μm, and after 30 seconds further 75 μM of each will beequilibrated. Thus 30 second bursts of excess ATP could be released in50 μM bursts to support burst of recombinase activity. Of course thechanging overall absolute concentrations of ADP and ATP are likely toaffect the reaction behaviour necessitating adjustments and possiblyslightly larger pulses of ATP may need to be release at each round.Nevertheless it is apparent that with a starting concentration ofcaged-ATP of 3 mM (similar to that used in our earlier experiments) andpulses of 50 μM, it is possible to support 60 independent pulses. Evenif the burst size needs to increase progressively to account for theoverall increase in ADP concentration it is fairly likely that 30 cyclescan be accommodated. Furthermore the ADP may be removed from thereaction by inclusion of ADP metabolising enzymes in the reaction. Thesecould include hydrolysis of ADP, or alternatively regeneration of ADP toATP and a constant source of alternative consuming ATP activity such asNADH generation.

Caged ATP is readily commercially available, and recently cheap lowpower devices that might be used to drive uncaging have becomeavailable. Advances in light-emitting diodes (LEDs) have lead to thedevelopment of small cheap low power sources to generate light ofwavelength 365 nm. We envision integration of such devices into lowpower heated cells, which are portable and battery-operated.Alternatively brief periods of recombinase activity could be generatedsimply by repeated addition of small volumes of additional ATP, or byusing an oscillator system, such as the phosophofructokinase oscillator.

RPA Reaction Control Mediated by Use of Asymmetric Primers

One-sided RPA reactions can be driven from a single primer target site.In the absence of a facing primer, such reactions will generatesingle-stranded DNA. We have found that oligonucleotides possessing a 3′locked nucleic acid (LNA) [Di Giusto D A, King G C (2004) Strongpositional preference in the interaction of LNA oligonucleotides withDNA polymerase and proofreading exonuclease activities: implications forgenotyping assays. Nucl Acids Res 32(3): e32]nucleotide, cannot serveras primers for recombinase-mediated elongation by certain polymerases,such as the Bacillus stearothermophilus and Bacillus subtilis polymeraseI, but do serve as primers for other polymerase such as the E. coliKlenow fragment consistent with prior data showing that such 3′LNAcapped primers can serve as primers in polymerase extension reactionswhen they hybridise to target single-stranded DNAs. We have also foundthat not all polymerases seem to be able to initiate from recombinationintermediates, perhaps reflecting that for some polymerases longerstretches of single-stranded DNA with bound primers are required. Forexample we have found that phi-29 polymerase is unable to initiatesynthesis from a recombination intermediate. We also have evidence thatit can however synthesise from a 3′LNA-capped primer. Finally consistentwith published reports, we have found that for a given recombinase,there is a minimal primer length required for recombinase-assistedstrand-exchange to occur efficiently. Specifically oligonucleotidesshorter than 27-30 base pairs are poor substrates for uvsX. Neverthelessprimers as short as 20-27 nucleotides may be perfectly adequate tosupport hybridisation-mediated priming. Thus by combining a longer and ashorter primer one can bias RPA reactions to use one primer forinvasion-driven elongation and the other for hybridisation-drivenelongation.

Taken together these facts suggest a variety of configurations in whichreactions can be assembled in a way that synthesis initiates from oneside, and only when it has passed the second site does opposingsynthesis begin. For example, an RPA reaction may be configured suchthat one primer is a normal oligonucleotide and the opposing primer is a3′LNA-capped oligonucleotide. In the same reaction a polymerase thatcannot use 3′LNA-capped oligonucleotides, but works from invasionstructures, is mixed with one that can use 3′LNA-capped oligonucleotidesbut works only from hybridisation structures. Recombinase-mediatedinvasion and extension from the normal primer will generatesingle-stranded DNA molecules, which can then serve as templates forsynthesis by the second polymerase when a 3′LNA-capped opposing primerhybridises to its target site. Alternatively a short second primer thatfunctions only in hybridisation would also ensure asymmetric primer use.Other configurations of oligonucleotide length and nature, withdifferent polymerases, may be used to generate the desired effect. Thusthe use of asymmetric primers should resolve any interference fromreplication fork collision; however, it may be necessary also to controlrecombinase activity to avoid re-invasion of single-stranded DNAsdisplaced by synthesis from the normal primer.

The combination of asymmetric primers and control of ATP levels mayprovide conditions sufficient to amplify long (>10 kb) DNAs. One otherfactor that may affect the efficiency of amplification of long DNAs isinterference from other template DNAs, other non-template DNA, andproduct of the RPA reaction itself such as displaced single-strandedDNAs. In addition to the possibility that recombinase may associate witha displaced single stranded DNA and mediate an invasion reaction ontotemplate DNA, there is also the possibility that single stranded DNAsand other non-template DNAs from the sample may hybridiseinappropriately with target template DNAs and interfere with efficientreplication. To avoid some of these difficulties, at least for the firstfew rounds of replication it would be helpful to spatially fix templateDNA and prevent association with other long DNAs. One convenient meansto achieve this is to assemble RPA reactions in a gel matrix, such as apolyacrylamide gel adjusted with an average pore size that will allowfree mobility of small RPA components, such as the enzymes and primers,but not allow free motility of long DNAs. Sample DNAs at low startingconcentration will be physically separated, unable to associate with oneanother, while smaller RPA components will remain relatively free toassociate with template molecules. Church and colleagues have describedusing polyacrylamide gels in this way for PCR and colleagues to producespatially isolated amplicons, resolvable in two dimensions, known aspolymerase colonies, or polonies [Mitra R, Church G (1999) In situlocalized amplification and contact replication of many individual DNAmolecules. Nucl Acids Res 27(24): e34i-vi.]. Typically, polonies areused to image amplicons from distinct templates and so are generated onmicroscope slides. For amplification of long DNAs, however, it would notbe necessary to resolve individual polonies, thus reactions could beassembled in any appropriate vessel.

The polony assay itself may benefit from using RPA instead of PCR. Thereare at least two difficulties with the use of PCR. Firstly, becausepolonies are usually generated on microscope slides, and glass is a poorconductor of heat, the times required for PCRs can be significantlylonger than for normal bulk phase PCR. This means that diffusion ofamplification product leads to fairly large polony sizes, incompatiblewith high density, high throughput assays. Secondly, because PCRrequires the thermal melting of template DNA, temperatures in excess of90° C. are required for extended periods. These high temperatures willincrease diffusion rates further increasing the average polony size. RPAwith its low constant temperature will overcome both of these problems.

Using the RPA Dynamic Recombination Environment to Permit Identificationof the Polymorphic Status of a Given Amplicon

It is widely demonstrated that the presence or absence of a definednucleic acid sequence can be determined by forming a hybrid between asample nucleic acid with a nucleic acid probe of previously determinednature, followed by an appropriate method of detecting such aninteraction. For example, microarrays are now widely used in which DNA,RNA or other backbone oligonucleotides are spatially separated andimmobilized to a support. The presence or absence of a homologoussequence in a sample is then determined by co-incubating the samplesunder appropriate buffer and temperature conditions that allows hybridsto form between the immobilized probe and sample nucleic acids. In thiscase both sample and probe are provided in a completely or partiallymelted status so that they are able to hybridize. Provided a label isincorporated into the sample then the interaction can be subsequentlyquantified.

Alternative approaches to forming sequence-specific hybrids have beendescribed in which one of the participating nucleic acids isdouble-stranded at the outset, and a three-stranded protein-containinghybrid is formed by the action of recombinase enzymes such as E. colirecA in the presence of non-hydrolysable nucleotide triphosphate analogs(see U.S. Pat. No. 5,460,941, and U.S. Pat. No. 5,223,414). Alsodescribed are alternative methods that employ recombinases but seek tostabilize protein-free recombination intermediates by formingfour-stranded structures (see U.S. Pat. No. 5,273,881). These approacheshowever differ from those described herein both in method and outcome asdescribed further below.

In many cases the process of hybrid formation is of sufficient fidelityto discriminate between the presence and absence of the target, andfurther to determine whether it is a perfect match or not. For example,short primers (e.g. 7 to 18 nucleotides in length) can discriminatebetween perfect and imperfect complements if hybridization conditionsare stringently controlled. However, if the nucleic acid is longer andthere are only small variations between a perfect hybrid and animperfect hybrid, for example a few nucleotides over a region of say onehundred or more, then it unlikely that a sufficiently great differencein efficiency of hybridization will exist between such a variant and aperfect match.

This invention concerns determining the polymorphic state of a samplenucleic acid by forming hybrids between the products of an amplificationreaction and previously synthesized and immobilized probe nucleic acidspresent at defined locations, and each individual location containing apure population of fragments representing one of the known repeatlengths present in the population.

DNA sequences amplified by the recombination polymerase amplification(RPA) method are ideal targets for such a hybrid-formation based assayfor the following reasons. Firstly, it is possible to configure the RPAmethod to generate mostly double-stranded DNA product, or mostlysingle-stranded DNA product, for example by altering the ratios ofamplification primers. Secondly, RPA reactions contain all of thenecessary components to permit the association of hybrids betweeninitially duplex DNA and single-stranded DNAs with no requirement tothermally melt the double-stranded DNA. Thus little additional samplehandling is required.

Other methods have been described which employ recombinases and the useof non-hydrolysable analogs of ATP, such as ATP-γ-S, to stabilizehybrids of related complementary nucleic acids in a poorly defined‘triplex’ intermediate involving the continued presence of therecombinase. Such an approach, however, will not work in this thecontext described here, because these highly stable structures are notdynamic and present significant resistance of reaction products toaccess by other agents. As a consequence use of recombinases withnon-hydrolysable ATP analogs leads to formation of extremely stablehybrids between DNAs containing a significant degree of mis-match, thusnot allowing efficiently discrimination between sequences. In contrast,the approach described here employs a dynamic system utilizing ATP orother hydrolysable analogs in which hybrids are readily destabilized aswell as generated. The net result is a large enrichment for interactionsof perfect complementary.

In a preferred embodiment, following the completion of an RPA reactionor concomitant with it, the reaction mixture is contacted to an array ofprobe molecules such that each amplified polymorphic DNA will in thefirst instance locate the correct immobilised probe nucleic acidcontaining exactly the same, or complementary, sequence. Should thisinitial hybrid-forming reaction not occur with sufficient efficiency orfidelity, as is likely with very similar sequences, then the dynamicnature of the recombinase-driven reaction should resolve mismatches.This is because imperfect hybrids will contain bubbles and non-duplexfeatures, which act to aid the reloading of recombinase onto thesehybrids and cause an increased rate of hybrid disruption when comparedto perfect matches. New duplex-forming events are permitted to occur andonly if perfect hybrids are formed do they become relatively resistantto further reaction. In practice, if all of the possible number of anSTR repeat were arrayed in order of repeat number, then at the end theremay result a gradient of hybrids formed which peaks at exact matches, isweaker on the direct flanks and absent further way. As the mis-matchbubbles increase in size, there is a progressively greater tendency forrecombinase to reload onto the single-stranded bubble region. (FIGS. 58and 59).

Additionally, if the dynamic recombinase system is not sufficientlyspecific to discern between perfect and imperfect hybrids, additionalhybrid-disrupting components can be included in the reaction. Suchagents include, but are not limited to, helicases, nucleases,recombinases, polymerases, and other DNA-binding agents. Varioushelicases and nucleases exist which selectively target specific forms ofDNA, or structures, such that they interact with and resolve mismatchesor bubbles, but not on perfect hybrids. For example, the PriA helicaseof E. coli interacts with regions in which single-stranded DNA isexposed adjacent to double-stranded DNA, as would occur in a mismatch inrepeat number that could occur with STR hybridizations, and subsequentlycan act to unwind DNA in a 3′ to 5′ direction. Such destabilizationwould permit recombinase to re-load onto these separated strandsallowing a new alternative hybrid to form instead. Over time such amechanism would enrich for hybrids only between the correct target andprobe. Similarly, the replication helicase of E. coli, DnaB, will loadonto single-stranded DNA and unwind duplex DNA, in a 5′-3′ direction,opposite of that of PriA. The dda helicase of bacteriophage T4 loadsonto single-stranded DNA. The dda helicase is so powerful it candisplace other DNA bound proteins in its path. The dda helicase has beenshown to disrupt very high affinity streptavidin-biotin interactions(when the biotin is on the 3′ end of the oligonucleotide), thus ddahelicase could be used to destabilize immobilized probe complexes on asurface (Byrd and Raney, 2004) (Morris and Raney, 1999).

Alternatively the E. coli RuvA and RuvB gene products form a helicasethat can encircle double-stranded DNA and drive branch points along DNA.In this manner a bubble could be ‘pushed’ to the end of the templatecausing sufficient destabilization to permit recombinase loading as wellas other event. Nucleases that would target imperfections in duplexcharacter would include, for example, S nuclease, which can nickdouble-stranded DNA at a mismatch or bubble. Other such nucleases withstructure-specific characteristics exist. Such nicks can serve toinitiate strand-displacing polymerase elongation. Alternatively if thenuclease cleaves the probe DNA then it is possible to have a chemical orenzymatic group released from the site of immobilization so that overtime a signal generated later remains only at the point at which perfecthybrids formed.

Thus by combining the necessary components it should be possible tocreate an environment in which perfect hybrids significantly dominateand imperfect sites are depleted, or are rendered undetectable. Key tothis approach is the establishment of a dynamic environment in whichhybrids can be formed, and disrupted such that the stability of perfectand imperfect hybrids allows sensitive discrimination of reactionproducts with differing polymorphic features. This environment isideally provided by the dynamic/stable recombinase system comprisinggp32, uvsX, uvsY, PEG compound, and an ATP regenerating system asdescribed.

The presence or absence of a productive hybrid, or the loss of a labelfrom an immobilization site (e.g. see nuclease approach above) can bemeasured by standard methods.

These include incubation of the reaction at the end-point with asubstrate for an enzyme immobilized on the DNA target or DNA probe.Other detection approaches are possible and described widely elsewhere.

The Dynamic Persistent Recombinase Environment for Broad Use inMolecular Techniques

We set about establishing the perfect environment for dynamicrecombinase activity with a mind to permitting massively geometricamplification of nucleic acids at low constant temperature. However theestablished environment might obviously be employed in combination withother enzymes, or indeed without them, in a variety of contexts toreplace classical hybridisation reactions in general. For examplemolecular cloning procedures might take advantage of the possibility ofrecombining large tracts of DNA with one another much more effectivelythan by thermal melting and annealing. Also many other enzymes thatmight be employed for in vitro molecular processes could benefit fromthe low temperature environment compatible with most mesophilic enzymes.Such enzymes would include, in addition to polymerases, helix-distortionrecognising nucleases (e.g. Si nuclease), FLAP endonucleases,restriction endonucleases, base modifying or removing enzymes, lyases,helicases, topoisomerases, ligases, reverse transcriptases, RNase Hactivity, resolvases, RNA polymerases, and any other enzyme that actson, or interacts with, nucleic acids. They could also involve otherenzymes pertinent to the in vitro system in addition to DNA metabolisingenzymes, such as alkaline phosphatase or horseradish peroxidase used indetection protocols. The combination of the unique properties of thestable dynamic recombination system with other enzymatic activitiesenables a potentially very large number of new methods and applications.

In addition to recognising the important impact of combining whollylow-temperature dynamic enzymatic hybridisation systems with otherenzyme systems, we have established sequence knowledge for the optimalassembly of highly active recombination using short oligonucleotideprimers on the basis of experiments using RPA. We have noted that veryactive primers were characterised by a particularly rich distribution ofpyrimidines. Guanosine residues, in contrast, appeared poorlyrepresented in the most active oligonucleotides that we have analysed,and earlier results suggested that when appended to the 5′ end ofoligonucleotides they might lower their activity. In this disclosure wereport the experimental results of dynamic monitoring of the reaction tostudy the affects of appending DNA sequences to the 5′ ends ofoligonucleotides. Appending sequences to the very 5′ extent of otherwiseidentical oligonucleotides has a significant effect on their activity inRPA. We suggest that these observations are best interpreted asreflecting the loading behaviour of the recombinase on single-stranded,and possibly duplex, DNA. They may also have roles in controllingdefined phasing of recombinases on ssDNAs.

As a starting point, we had come to note through kinetic studies (nonreal-time), and general observations, that some primer pairs werecapable of mediating more rapid DNA amplification than others.Particularly rapid primers were identified for the human STR markerCSF1PO, which appeared to have an average doubling time of roughly 30seconds (as estimated as average doublings per unit time from start todetectable product accumulation), and capable of generating detectablelevels of product within 15 minutes starting with a few thousand copiesof target (see FIG. 67). Other primer pairs typically took, taking 20-35minutes to achieve similar results. We sought to learn more about thesource of this variability (see FIG. 67, and data not shown).

Analysis of the DNA sequence of the CSF1PO primers revealed that theywere relatively rich in pyrimidines, and also were rather low inguanosine. This observation correlated with a piece of data arising froman earlier experiment in which we had investigated how appending astretch of cytosine or guanosine residues to the 5′ end ofoligonucleotides affected their behaviour in RPA. In FIG. 68 a real-timeamplification experiment is shown in which primers specific for theBacillus subtilis sporulation locus SpOB, referred to as J1 and K2, wereused to amplify a fragment from B. subtilis genomic DNA. In theexperiment shown in FIG. 67 we had noted that a stretch of guanosineshad ‘quietened’ the overall reaction, while a stretch of appendedcytosines had ‘noisened’ amplification reactions. We repeated thisexperiment and monitored the reaction in real-time, the findings shownin FIG. 68. The results were consistent with the notion that appendedbases affected, at the very least, the rate behaviour and presumablyactivity of primers. Primers J1 and K2, in which all bases match genomictarget, generate a product which is first detectable with SYBR green dyeat a little over 30 minutes in this experiment (faster kinetics wereobserved in later experiments, and variation may have arisen due tonon-optimal temperature ramping when using a conventional microplatereader, as occurred in these experiments). A sample with target DNA, andone without, were distinguishable in this experiment by virtue of asmall delay in product accumulation, and variant accumulation kinetics.Primers appended with additional cytosine residues, J1(C) and K2(C),were clearly capable of amplifying DNA significantly more quickly. Wenote however that the accumulation of (primer-derived) artifacts occurson a timescale similar to that with the target DNA, distinctly less-wellseparated than in the unappended primers. We deduce that C-tailedprimers are simply faster primers (although ‘noisier’ also in thiscase). Conversely accumulation kinetics for the primers appended with Gresidues was very poor, and no clearly identifiable expected end-productwas observed on gel electrophoresis even at 90 minutes.

Based on our results, and integrating these findings with publishedwork, lead us to speculate that sequence-composition might affectrecombinase-loading onto primers, and that pyrimidines might promotethis loading process. Published work is conflicting in this area; insome studies a case has been made for preference of recA binding torecombination processes for G and T residues which are enriched in E.coli recombination hotspots (Tracy and Kowalczykowski). Conversely,studies of recA loading dynamics performed using fluorescence anisotropydrew startlingly different conclusions (Bar-Ziv and Libchaber). In thiscase the barrier to recA nucleation (the slow phase of nucleoproteinfilament formation) was highly sensitive to composition, showing astrong favourable bias towards pyrimidines. These latter observationswould be more consistent with our observations. Indeed, as RPA tends toemploy rather short oligonucleotides, it becomes easy to see howimportant the recombinase nucleation event is in giving effectiveamplification behaviour. Not only is rapid nucleation highly desirableto permit acceptable levels of filament loading, but additionally itwill be critical that this occurs preferentially towards the very 5′ endof the filament. If the 5′ end is a poor substrate for nucleation, whilean internal sequence is rather good, there is a likelihood that mostfilaments will be only partially loaded, and as such fail to functionproperly in RPA. Such partially loaded filaments may be insufficientlyloaded to hydrolyse ATP properly, unable to efficiently undergo strandexchange, and be effectively drawn away from an active pool offilaments. Even worse, they may ‘poison’ the reaction by forminghomology-searching complexes with DNA, but, being unable to undergo thenormal dynamic behaviour (if they are below necessary length to promotehydrolysis), lock up target DNA in unproductive complexes, as occurswhen ATP-γ-S is employed.

Despite our early results being consistent with the notion thatpyrimidines are good nucleation sites, and the attractive possibilitythat this alone accounts for the observed variation, we must point outthat neither of the above assays is truly equivalent to the experimentalsystem that we are employing. Filament loading, unloading, hydrolysisbehaviour and so on, are all relevant in concert in the amplificationsituation, and multiple factors could be at play. Also, it likely thatrecombinase phasing could play a role in determining the activity ofprimers, and that this established by preferential nucleation atparticular locations, a phenomenon reported earlier for recA (Volodin etal.; Volodin and Camerini-Otero). In this case nucleation would occurpreferentially at the 5′ end, establish a committed phase to the entirefilament, and the filament length would need to have a perfect length toplace the last protein over the 3′ end in the most desirable manned. Inaddition to the further complexities associated with the amplificationsituation as described, these earlier studies were all performed with E.coli RecA protein, and not T4 UvsX protein, and as these proteins aresignificantly diverged at the primary sequence level, it may not bepossible to extrapolate conclusions from RecA studies to the UvsXprotein. Nonetheless, realising the importance of sequence composition,and in particular 5′ sequence composition, we have begun to analyse inmore depth which sequences are most effective for appending to the very5′ end, i.e. we seek to determine idealized ‘landing sites’ for therecombinase in this system, and/or those that are associated with otherbeneficial properties for amplification and in vitro processes ingeneral. Yet further, we anticipate that phasing of recombinases mayplay a significant role in overall activity, and that this will beinfluenced by composition and precise length of the oligonucleotide.

We investigated whether appending a stretch of Thymine residues, or amixture of cytosine and thymines (pyrimidines) would be as effective asa stretch of cytosine residues in stimulating greater primary activity.The results of this analysis are shown in FIG. 69. Interestingly,appending a run of Thymines to the very 5′ end of the oligonucleotidesdid not, in this experiment, improve the amplification rate behaviour.In fact rather to the contrary combining primers with appended thyminesgave very poor amplification behaviour in this experiment. We alsotested primers in which the appended sequence was a mixture of cytosineand thymine, as indicated. The behaviour of these primers in variouscombinations was rather more variable and complex. For example we notedthe presence of additional bands consistent, but not proven to be,single-stranded equivalents of the product. Careful analysis shows thatin different combinations the migration behaviour of this possiblesingle-stranded band was either of one type, or a second type, and thatthis correlated with the primer used. In brief, we suspect that in thesereactions one, or other, primer was more active leading to developmentof asymmetry in the reaction and single-stranded DNA accumulation notseen with the parent, unappended, oligonucleotides. It was difficult torationalise the results, but we can conclude that in this experimentstretches of thymine were not effective, nor were mixed polymers veryeffective despite a slight tendency to appear more active if morecytosines were present.

In our next experiment we appended yet more residues to the end ofoligonucleotides and compared their behaviour in a real-time analysis.Once again there was variability between primers, and in this experimentwe included as a control each primer incubated on its own. Interestinglywe noted that, at the very low concentrations of target used here (1copy per microliter start density), noise generated by single primersappeared in a timeframe similar to the accumulation of product DNAobserved when primer pairs were used. Furthermore, this is most obviousfor the oligonucleotide K2(C), studied previously, and responsible forvery rapid amplification when combined with J1 oligonucleotides. Thissuggests that monitoring oligonucleotides individually for their rate ofnoise generation is of utility in determining their overall activity.Finally, and puzzlingly, an oligonucleotide just one base pair shorterthan K2(C), oligonucleotide 9 in the series in FIG. 70, lacked thisstrong and rapid amplification behaviour. While the oligonucleotidesused in this study were not HPLC purified, we nonetheless assume thatmost of the oligonucleotides are the full length form (based on gelpictures supplied by the manufacturer). Taken at face value, thissurprising observation must be taken to reflect that either the numberof C residues, changing from 5 to 6, at the 5′ end is a point ofsignificant transition for some structural feature of thisoligonucleotide, or it could be taken to reflect the additionalinfluence of some phasing behaviour. In this latter case we wouldspeculate that 5′ sequences strongly influence the phasing anddeposition of the first UvsX monomer, and that this phasing ismaintained all the way down to the 3′ end. Presumably whether or not themost 3′ UvsX monomer sits perfectly over the end of the oligonucleotide,or there is slightly too many or too few base pairs to permit each UvsXmonomer to bind to its maximal number of backbone residues, may wellinfluence the likelihood that recombination proceeds efficiently tocompletion. It may also influence the background, as if there are a few‘spare’ bases popping out of the 3′ end this might well promoterenhanced primer noise.

In conclusion, we have shown here how critical the length and sequencecomposition of oligonucleotides may be to elicit the best performance inRPA, and other recombinase-driven processes. Further, we formallydemonstrate that non-target sequences can be appended to the 5′ ends ofoligonucleotides to effect regulation of their activity in RPA.

In addition to DNA amplification, a recombinase system includinghydrolysable ATP analogs, and components to ensure high loading of DNAmolecules in this situation, could be employed as a replacement forother described hybridisation approaches.

Contamination Control in RPA Reactions

As RPA is a very sensitive detection method we have encountered problemswith carry-over contamination seen with other ultra-sensitiveamplification protocols such as PCR. We show here that dUTP can be usedto partially or wholly replace dTTP in RPA reactions, thus offering asimple way to distinguish the products of previous RPA reactions frombona fide sample targets. As heat treating RPA reactions at theirinitiation with a mind to inactivating dUTP deglycosylase (as occurs inPCR protocols with contamination control) is not desirable, we suggestas alternative approach in which deglycosylase inhibitors are mixed withreactions to permit initiation (see FIGS. 61 and 62).

Reverse-Transcription RPA

In circumstances in which detection of the presence of specific RNAmolecules is desired it is possible to use RPA to detect them providingthat the RNA is first converted to a DNA form using reversetranscriptase activity. Most convenient would be if reversetranscription and RPA could all be performed in a single homogeneousenvironment. We show that reverse transcription RPA reactions (RT-RPA)can be performed in a single tube environment by including reversetranscriptase in the reaction environment, and with other minormodifications (see FIG. 60).

Straightforward Detection and Assessment of Amplification ReactionsUsing Lateral Flow Membranes.

Features of RPA make it ideally suited to configuring portableeasy-access diagnostic intergrated products. In one set of casesassessment of whether a specific DNA amplification reaction has occurredor not, with only a moderate need for quantitative analysis, can beperformed by simple formats to assess whether two labelled primers havebecome physically associated within an amplicon. We here show that thissimple idea is effective, and in particular that the widely-employedtechnology of lateral-flow strip systems are ideal to perform this role.RPA reactions can be mixed directly with sample running buffer for thesesystems, and the presence of amplicon determined within several minutes(see FIG. 63).

Compatibility of Crude Preparations of Biological Samples with RPA

We here show that simple lysates of blood can be used directly in RPAreactions. This offers the possibility that the format of diagnosticproducts containing the RPA system may require only trivial treatment ofsamples prior to direct addition to RPA reactions (see FIG. 64).

Behaviour of Fluorescent Probes in the Stable, Persistent, DynamicRecombination Environment

We here show that the presence of the dynamic recombination environmentused in RPA alters the behaviour of fluorescent probes relative toequivalent environments in other techniques such as PCR. Mostparticularly if dual-labeled probes containing fluorophores andquenchers are to be used, the number of bases separating the two groupsin the oligonucleotide must be less, presumably because the saturatingDNA binding proteins stretch the probes relative to both their state inB-form duplex DNA, as well as the random coil that exists foroligonucleotides in free solution (FIG. 73). Furthermore we haveidentified key enzymes that may be employed to specifically processduplex hybrids between such probes and their target DNA's. Theseapproaches teach the approach by which real-time ‘third’ probestrategies musts be configured with RPA, and how that differs to thewell-established approaches in PCR such as the ‘Taqman’ approach,molecular beacons, and the like. In particular we have determined thatthe ‘Taqman’ approach cannot be employed in RPA presumably because the5′ nuclease associated with E. coli Pol I like enzymes has FLAPendonuclease activity which inhibits RPA reactions.

REFERENCES

-   Adams, D. E., Tsaneva, I. R. and West, S. C. (1994). Dissociation of    RecA filaments from duplex DNA by the RuvA and RuvB DNA repair    proteins. Proc Natl Acad Sci USA 91, 9901-5.-   Alexseyev, A. A., Bakhlanova, I. V., Zaitsev, E. N. and    Lanzov, V. A. (1996). Genetic characteristics of new RecA mutants of    Escherichia coli K-12. J Bacteriol 178, 2018-24.-   Bar-Ziv R, Libchaber A. (2001). Effects of DNA sequence and    structure on binding of RecA to single-stranded DNA. PNAS USA July    31; 98(16):9068-73-   Baumann, P., Benson, F. E., Hajibagheri, N. and West, S. C. (1997).    Purification of human Rad51 protein by selective spermidine    precipitation. Mutat Res 384, 65-72.-   Benkovic, S. J., Valentine, A. M. and Salinas, F. (2001).    Replisome-mediated DNA replication. Annu Rev Biochem 70, 181-208.-   Bennett, R. L. and Holloman, W. K. (2001). A RecA homologue in    Ustilago maydis that is distinct and evolutionarily distant from    Rad51 actively promotes DNA pairing reactions in the absence of    auxiliary factors. Biochemistry 40, 2942-53.-   Better, M. and Helinski, D. R. (1983). Isolation and    characterization of the RecA gene of Rhizobium meliloti. J Bacteriol    155, 311-6.-   Bianco, P. R. and Weinstock, G. M. (1996). Interaction of the RecA    protein of Escherichia coli with single-stranded    oligodeoxyribonucleotides. Nucleic Acids Research December 15;    24(24):4933-9-   Bork, J. M., Cox, M. M. and Inman, R. B. (2001). The RecOR proteins    modulate RecA protein function at 5′ ends of single-stranded DNA.    EMBO J 20, 7313-22.-   Bork, Cox and Inman J Biol Chem. 2001 Dec. 7; 276(49):45740-3-   Butler B C, Hanchett R H, Rafailov H, MacDonald G (2002)    Investigating Structural Changes Induced By Nucleotide Binding to    RecA Using Difference FTIR. Biophys J 82(4): 2198-2210-   Byrd A K, Raney K D (2004). Protein displacement by an assembly of    helicase molecules aligned along single-stranded DNA. Nat Struct Mol    Biol. June; 11(6):531-8-   Cox, M. M., Goodman, M. F., Kreuzer, K. N., Sherratt, D. J.,    Sandler, S. J. and Marians, K. J. (2000). The importance of    repairing stalled replication forks. Nature 404, 37-41.-   Cox, M. M., McEntee, K. and Lehman, I. R. (1981). A simple and rapid    procedure for the large scale purification of the RecA protein of    Escherichia coli. J Biol Chem 256, 4676-8.-   Cromie, G. A. and Leach, D. R. (2000). Control of crossing over. Mol    Cell 6, 815-26.-   Dillingham, M. S, and Kowalczykowski, S. C. (2001). A step backward    in advancing DNA replication: rescue of stalled replication forks by    RecG. Mol Cell 8, 734-6.-   Eggler, Lusetti and Cox, J Biol Chem. 2003 May 2; 278(18):16389-96-   Eggleston, A. K. and West, S. C. (2000). Cleavage of holiday    junctions by the Escherichia coli RuvABC complex. J Biol Chem 275,    26467-76.-   Elias-Arnanz M, Salas M. EMBO J. 1997 Sep. 15; 16(18):5775-83-   Ferrari et al., J Mol Biol. 1994 Feb. 11; 236(1):106-23-   Formosa T, Alberts B M. J Biol Chem. 1986 May 5; 261(13):6107-18-   Formosa T, Alberts B M. Cell. 1986 Dec. 5; 47(5):793-806. DNA    synthesis dependant on genetic recombination: characterization of a    reaction catalyzed by purified bacteriophage T4 proteins.-   Glover, B. P. and McHenry, C. S. (2001). The DNA polymerase III    holoenzyme: an asymmetric dimeric replicative complex with leading    and lagging strand polymerases. Cell 105, 925-34.-   Goodman, H. J., Parker, J. R., Southern, J. A. and Woods, D. R.    (1987). Cloning and expression in Escherichia coli of a RecA-like    gene from Bacteroides fragilis. Gene 58, 265-71.-   Hacker K J, Alberts B M. J Biol Chem. 1992 Oct. 15; 267(29):20674-81-   Harris L D, Griffith J. J Biol Chem. 1987 Jul. 5; 262(19):9285-92-   Heid C A, Stevens J, Livak K J, Williams P M. Real time quantitative    PCR. Genome Res. 1996 October; 6(10):986-94.-   Heyer, W. D. and Kolodner, R. D. (1989). Purification and    characterization of a protein from Saccharomyces cerevisiae that    binds tightly to single-stranded DNA and stimulates a cognate strand    exchange protein. Biochemistry 28, 2856-62.-   Hickson, I. D., Gordon, R. L., Tonikinson, A. E. and Emmerson, P. T.    (1981). A temperature sensitive RecA protein of Escherichia coli.    Mol Gen Genet 184, 68-72.-   Hsieh, P., Camerini-Otero, C. S, and Camerini-Otero, R. D. (1992).    The synapsis event in the homologous pairing of DNAs: RecA    recognizes and pairs less than one helical repeat of DNA. Proc Natl    Acad Sci USA 89, 6492-6.-   Hsieh P, Camerini-Otero C S, Camerini-Otero R D. Proc Natl Acad Sci    USA. 1992 Jul. 15; 89(14):6492-6-   Kato, R. and Kuramitsu, S. (1993). RecA protein from an extremely    thermophilic bacterium, Thermus thermophilus HB8. J Biochem (Tokyo)    114, 926-9.-   Katz F S, Bryant F R. Biochemistry. 2001 Sep. 18; 40(37):11082-9    Kelman, Z. and O'Donnell, M. (1995). DNA polymerase III holoenzyme:    structure and function of a chromosomal replicating machine. Annu    Rev Biochem 64, 171-200.-   Komori, K., Miyata, T., DiRuggiero, J., Holley-Shanks, R., Hayashi,    I., Cann, I. K., Mayanagi, K., Shinagawa, H. and Ishino, Y. (2000).    Both RadA and RadB are involved in homologous recombination in    Pyrococcus furiosus. J Biol Chem 275, 33782-90.-   Kornberg, A. and Baker, T. A. (1992). DNA Replication. New    York: W. H. Freeman and Company. Kuil et al., Biophys Chem. 1988    December; 32(2-3):211-27-   Kowalczykowski S C, Lonberg N, Newport J W, Paul L S, von Hippel P    H., Biophys J. 1980 October; 32(1):403-18.-   Kuramitsu, S., Hamaguchi, K., Ogawa, T. and Ogawa, H. (1981). A    large-scale preparation and some physicochemical properties of RecA    protein. J Biochem (Tokyo) 90, 1033-45.-   Kurumizaka, H., Ikawa, S., Ikeya, T., Ogawa, T. and Shibata, T.    (1994). A chimeric RecA protein exhibits altered double-stranded DNA    binding. J Biol Chem 269, 3068-75.-   Layery P E, Kowalczykowski S C. J Biol Chem. 1992 May 5;    267(13):9307-14-   Liu, J., Nurse, P. and Marians, K. J. (1996). The ordered assembly    of the phiX174-type primosome. III. PriB facilitates complex    formation between PriA and DnaT. J Biol Chem 271, 15656-61.-   Lohman and Ferrari, Annu Rev Biochem. 1994; 63:527-70-   Lovett, C. M., Jr. and Roberts, J. W. (1985). Purification of a RecA    protein analogue from Bacillus subtilis. J Biol Chem 260, 3305-13.-   Maeshima, K., Morimatsu, K. and Horii, T. (1996). Purification and    characterization of XRad5 1.1 protein, Xenopus RAD51 homologue:    recombinant XRad5 1.1 promotes strand exchange reaction. Genes Cells    1, 1057-68.-   Marians, K. J. (1992). Prokaryotic DNA replication. Annu Rev Biochem    61, 673-719.-   Marians, K. J. (1999). PriA: at the crossroads of DNA replication    and recombination. Prog Nucleic Acid Res Mol Biol 63, 39-67.-   Mazin, A. V. and Kowalczykowski, S. C. (1998). The function of the    secondary DNA-binding site of RecA protein during DNA strand    exchange. EMBO J 17, 1161-8.-   McGlynn, P. and Lloyd, R. G. (1999). RecG helicase activity at    three- and four-strand DNA structures. Nucleic Acids Res 27,    3049-56.-   McGlynn, P., Mahdi, A. A. and Lloyd, R. G. (2000). Characterisation    of the catalytically active form of RecG helicase. Nucleic Acids Res    28, 2324-32.-   Mitra, R., Church, G. (1999). In situ localized amplification and    contact replication of many individual DNA molecules. Nucl Acids Res    27(24): e34i-vi-   Morel, P., Chemy, D., Ehrlich, S. D. and Cassuto, E. (1997).    Recombination-dependent repair of DNA double-strand breaks with    purified proteins from Escherichia coli. J Biol Chem 272, 17091-6.-   Morrical S W, Wong M L, Alberts B M. J Biol Chem. 1991 Jul. 25;    266(21):14031-8. Amplification of snapback DNA synthesis reactions    by the uvsX recombinase of bacteriophage T4.-   Morrical and Alberts J Biol Chem. 1990 Sep. 5; 265(25):15096-103.    The UvsY protein of bacteriophage T4 modulates    recombination-dependant DNA synthesis in vitro.-   Morris P D, and Raney K D. (1999). DNA helicases displace    streptavidin from biotin-labeled oligonucleotides. Biochemistry    April 20; 38(16):5164-71-   Ng, J. Y. and Marians, K. J. (1996a). The ordered assembly of the    phiX174-type primosome. I. Isolation and identification of    intermediate protein-DNA complexes. J Biol Chem 271, 15642-8.-   Ng, J. Y. and Marians, K. J. (1996b). The ordered assembly of the    phiX174-type primosome. II. Preservation of primosome composition    from assembly through replication. J Biol Chem 271, 15649-55.-   Paulus, B. F. and Bryant, F. R. (1997). Time-dependent inhibition of    RecA protein-catalyzed ATP hydrolysis by ATP-γS: evidence for a    rate-determining isomerization of the RecA-ssDNA complex.    Biochemistry 36, 7832-8.-   Pham, P., Bertram, J. G., O'Donnell, M., Woodgate, R. and    Goodman, M. F. (2001). A model for SOS-lesion-targeted mutations in    Escherichia coli. Nature 409, 366-70.-   Pierre, A. and Paoletti, C. (1983). Purification and    characterization of RecA protein from salmonella typhimurium. J Biol    Chem 258, 2870-4.-   Rashid, N., Morikawa, M., Kanaya, S., Atomi, H. and Imanaka, T.    (2001). RecA/RadS 1 homolog from Thermococcus kodakaraensis KODI.    Methods Enzymol 334, 261-70.-   Reddy, Weitzel and Von Hippel, Proc Natl Acad Sci USA. 1993 Apr. 15;    90(8):3211-5-   Riddles P W, Lehman I R. J Biol Chem. 1985 Jan. 10; 260(1):170-3-   Rosselli, W. and Stasiak, A. (1990). Energetics of RecA-mediated    recombination reactions. Without ATP hydrolysis RecA can mediate    polar strand exchange but is unable to recycle. J Mol Biol 216,    335-52.-   Salinas F, Jiang H, Kodadek T. J Biol Chem. 1995 Mar. 10;    270(10):5181-6. Homology dependence of UvsX protein-catalyzed joint    molecule formation.-   Scheerhagen et al., J Biomol Struct Dyn. 1986 April; 3(5):887-98-   Shan, Q., Bork, J. M., Webb, B. L., Inman, R. B. and Cox, M. M.    (1997). RecA protein filaments: end-dependent dissociation from    ssDNA and stabilization by RecO and RecR proteins. J Mol Biol 265,    519-40.-   Singleton, M. R., Scaife, S, and Wigley, D. B. (2001). Structural    analysis of DNA replication fork reversal by RecG. Cell 107, 79-89.-   Spies, M., Kil, Y., Masui, R., Kato, R., Kujo, C., Ohshima, T.,    Kuramitsu, S. and Lanzov, V. (2000). The RadA protein from a    hyperthermophilic archaeon Pyrobaculum islandicum is a DNA-dependent    ATPase that exhibits two disparate catalytic modes, with a    transition temperature at 75 degrees C. Eur J Biochem 267, 1125-37.-   Steffen, S. E. and Bryant, F. R. (2000). Purification and    characterization of the RecA protein from Streptococcus pneumoniae.    Arch Biochem Biophys 382, 303-9.-   Tang, M., Pham, P., Shen, X., Taylor, J. S., O'Donnell, M.,    Woodgate, R. and Goodman, M. F. (2000). Roles of E. coli DNA    polymerases IV and V in lesion-targeted and untargeted SOS    mutagenesis. Nature 404, 1014-8.-   Tissier, A. F., Lopez, M. F. and Signer, E. R. (1995). Purification    and characterization of a DNA strand transferase from broccoli.    Plant Physiol 108, 379-86.-   Tracy R B, Kowalczykowski S C (1996). In vitro selection of    preferred DNA pairing sequences by the Escherichia coli recA    protein. Genes Dev. August 1; 10(15):1890-903.-   Vester B, Wengel J., Biochemistry. 2004 Oct. 26;    43(42):13233-41Villemain, et al. J Biol Chem. 2000 Oct. 6;    275(40):31496-504-   Volodin A A, Camerini-Otero R D (2002). Influence of DNA sequence on    the positioning of RecA monomers in RecA-DNA cofilaments. J Biol    Chem. January 11; 277(2):1614-8.-   Volodin A A, Smirnova H A, Bocharova T N, Camerini-Otero R D (2003).    Phasing of RecA monomers on quasi-random DNA sequence. FEBS Lett.    July 10; 546(2-3):203-8-   Webb, B. L., Cox, M. M. and Inman, R. B. (1995). An interaction    between the Escherichia coli RecF and RecR proteins dependent on ATP    and double-stranded DNA. J Biol Chem 270, 31397-404.-   Webb, B. L., Cox, M. M. and Inman, R. B. (1997). Recombinational DNA    repair: the RecF and RecR proteins limit the extension of RecA    filaments beyond single-strand DNA gaps. Cell 91, 347-56.-   Webb, B. L., Cox, M. M. and Inman, R. B. (1999). ATP hydrolysis and    DNA binding by the Escherichia coli RecF protein. J Biol Chem 274,    15367-74.-   West, S. C., Countryman, J. K. and Howard-Flanders, P. (1983).    Purification and properties of the RecA protein of Proteus    mirabilis. Comparison with Escherichia coli RecA protein;    specificity of interaction with single strand binding protein. J    Biol Chem 258, 4648-54.-   Wetmur, J. G., Wong, D. M., Ortiz, B., Tong, J., Reichert, F. and    Gelfand, D. H. (1994). Cloning, sequencing, and expression of RecA    proteins from three distantly related thermophilic eubacteria. J    Biol Chem 269, 25928-35.-   Wittwer C T, Hermann M., Moss A A, and Rasmussen R P. Continuous    fluorescence monitoring of rapid cycle DNA amplification.    Biotechniques. 1997 January; 22(1):130-1, 134-8-   Wittwer C. T., Ririe K M, Andrew R V, David D A, Gundry R A, Balis    U J. The LightCycler: a microvolume multisample fluorimeter with    rapid temperature control.-   Biotechniques. 1997 January; 22(1):176-81 Xu L, Marians K J. J Biol    Chem. 2002 Apr. 19; 277(16):14321-8.

EXAMPLES

As shown herein, we have developed an in vitro DNA amplification systemthat couples recombinase-driven sequence targeting withstrand-displacement synthesis. This permits DNA amplification withoutglobal thermal, chemical, or enzymatic template melting. Reactions aresensitive, specific and operate at 37° C. with no pre-treatment ofsample DNA. As much as 1012-fold amplification is observed within1-1^(1/2) hours. Less than 10 copies of a given target DNA can bedetected in a complex sample with a simple single-step reaction. Thismethod is an ideal alternative to PCR for a variety of applications andwill enable highly portable DNA diagnostic systems.

The examples are presented in order to more fully illustrate thepreferred embodiments of the invention. These examples should in no waybe construed as limiting the scope of the invention, as encompassed bythe appended claims.

Example 1 An Example of a Leading Strand Recombinase-PolymeraseAmplification (lsRPA)

DNA sequences can be amplified using leading strand synthesis accordingto the Recombinase-Polymerase amplification (RPA) method depicted inFIG. 1. FIG. 1 shows RecA/primer loading. Prior to the addition oftemplate DNA and/or Polymerase, RecA and SSB will compete for binding tosingle-stranded oligonucleotide primers. In the presence of a RecR andRecO, RecA is selectively stabilized onto the single-stranded primersforming RecA nucleoprotein filaments in a complex with RecO and RecR.This complex is competent to invade double-stranded DNA to form a D-loopat sites homologous to the oligonucleotide primers. Alternatively, RecA,RecO and RecR can be pre-loaded onto oligonucleotide primers prior tothe introduction of SSB to the reaction mixture.

The following details the likely composition of an RPA reactionassembled with E. coli recA and E. coli recO and recR stabilizingagents:

D-LOOP FORMATION/RESOLUTION COMPONENTS: Component Concentration RecA 20μM Single-stranded oligonucleotide primers 0.25 μM ATP 3 mM RecF 0.1 μMRecO 0.13 μM RecR 0.5 μM Single-stranded Binding protein (SSB) 1 to 10μM DNA polymerase V 5 units

POLYMERASE/HELICASE/RESOLVASE MIX Component Concentration DNA Polymerase5 units RuvA 0.5 μM RuvB 0.5 μM RuvC 0.5 μM RecG 10 nM

REACTION BUFFER Component Concentration MgCl2 2 2 to 10 mM TrisHCl pH7.2 50 mM DTT 0 to 10 mM KCl 0 to 50 mM Deoxyribonucleotidetriphosphates 0.2 mM Bovine serum albumin (BSA) 0 to 10 μg per ml

The reaction is assembled so that the final concentration satisfies theD-Loop Formation/Resolution Components, Polymerase/Helicase/ResolvaseMix, and Reaction Buffer with the DNA polymerase and/or template addedlast if necessary. For example, a 2.times. concentrated solution ofD-Loop Formation/Resolution Components and of thePolymerase/Helicase/Resolvase Mix may be made in 1× reaction buffer. Thereaction may be initiated by mixing an equal volume of each of the twocomponents (each in 1× reaction buffer). Optionally, and as statedabove, the DNA polymerase or template (target DNA) may be added last.The reaction is incubated for a sufficient time of until the reactantsare exhausted. Typical incubation times would range from 1 hour, 2hours, 3 hours, 5 hours, 10 hours or overnight (about 16 hours). UnlikePCR, which requires small volumes for rapid temperature change, there isno limit to the reaction volume of RPA. Reaction volumes of 25 μl, 50μl, 100 μl, 1 ml, 10 ml and 100 ml or larger may be performed in onevessel. Incubation temperature may be a typical laboratory temperaturesuch as 25° C., 30° C., or 37° C.

Prior to the addition of template DNA and/or Polymerase, recombinase andSSB will compete for binding to single-stranded oligonucleotide primers.In the presence of a RecR and RecO, RecA is selectively stabilized ontothe single-stranded primers forming RecA nucleoprotein filaments in acomplex with RecO and RecR. This complex is competent to invadedouble-stranded DNA to form a D-loop at sites homologous to theoligonucleotide primers. Alternatively, RecA, RecO, and RecR can bepre-loaded onto oligonucleotide primers prior to the introduction of SSBto the reaction mixture (FIG. 1).

The invading strands will be extended by the polymerase in a 5′ to 3′direction. As D-loops are formed and synthesis proceeds, displacedsingle stranded DNA becomes coated with SSB. RecA release fromdouble-stranded DNA can occur via ATP hydrolysis in a 5′ to 3′ directionor as a result of helicase/resolvase or polymerase activity (FIG. 2A,B). New rounds of invasion/synthesis will continuously occur. The thirdround of strand-invasion/synthesis will release discrete productsreleased whose ends correspond to the two facing primer sites. Thesefragments will soon become the dominant reaction product and willaccumulate to high levels. As each synthetic complex processes to theend of the template RecA protein is displaced either by polymeraseactivity or by the activity of helicases, such as RuvAB or resolvases,such as RuvC. Once primers, ATP, deoxynucleoside triphosphates, or anyother limiting component is exhausted, the reaction will stop.

The inclusion of temperature-sensitive recombinase mutants will allowthe controlled initiation of DNA synthesis. In such a situation, theinitiation reaction is performed at 25 to 37° C. permitting theformation of D-loops. Elongation reactions are performed at 42° C.,which is non-permissive for RecA mediated double-strand invasion. Thenumber of cycles will determine the amount of reaction product. Extendedelongation phases will permit the amplification of extremely long DNAswithout interference of re-invasion.

Example 2 Nested RPA

The RPA reaction is performed as described in Example 1. A fraction ofone tenth ( 1/10) and one hundredth ( 1/100) of the reaction is removedand used in place of the DNA template in a second round of RPA. LsRPA,leading/lagging RPA, and combinations thereof may be used for nestedRPA.

Example 3 Simultaneous Leading and Lagging Strand Recombinase-PolymeraseAmplification

DNA sequences can be amplified using simultaneous leading and laggingstrand synthesis according to the Recombinase-Polymerase amplification(RPA) method depicted in FIG. 2. This figure specifically illustrateslsRPA. FIG. 2A shows that RecA/primer nucleoprotein filaments invadedouble stranded template DNA preferentially associating with homologoustarget sites. As D-loops are formed and synthesis proceeds, displacedsingle stranded DNA becomes coated with SSB (FIG. 2A). RecA release fromdouble-stranded DNA can occur via ATP hydrolysis in a 5′-3′ direction oras a result of helicase/resolvase or polymerase activity (FIG. 2A). Assynthesis continues (FIG. 2B), polymerases encounter SSB bound,displaced single-stranded template. Double-stranded target sites arere-invaded by RecA/primer nucleoprotein filaments. Subsequent rounds oflsRPA proceed from re-invaded sites (FIG. 2B).

The following details likely components of a replisome-mediatedamplification utilizing components from E. coli. A reaction is assembledwith the following composition:

D-LOOP FORMATION/RESOLUTION COMPONENTS Component Concentration RecA 20μM Single-stranded oligonucleotide primers 0.25 μM ATP 3 mM RecF 0.1 μMRecO 0.13 μM RecR 0.5 μM Single-stranded Binding protein (SSB) 1 to 10μM DNA polymerase V 5 units

HELICASE/RESOLVASE MIX Component Concentration RuvA 0.5 μM RuvB 0.5 μMRuvC 0.5 μM RecG 10 nM

PRIMOSOME COMPLEX Component Concentration PriA 20 nM PriB 20 nM DnaT 100nM DnaB 100 nM DnaC 200 nM DnaG 200 nM

DNA POLYMERASE III HOLOENZYME COMPLEX Component Concentration B-Clamp 2μM DnaX Clamp Loader 500 nM Polymerase Core Complex 500 nM

LAGGING STRAND MIX Component Concentration DNA polymerase I 5 units DNAligase 2 units Polymerase Core Complex 500 nM

REACTION BUFFER Component Concentration MgCl₂ 2 to 10 mM TrisHCl pH 7.210 to 60 mM DTT 0 to 10 mM KCl 0 to 50 mM Deoxyribonucleotidetriphosphates 0.2 to 0.4 mM Bovine serum albumin (BSA) 0 to 10 μg per ml

The reaction is assembled so that the final concentration of all thereagents is as listed above. Thus, for example, a 5 fold concentratedsolution of each of the components (D-loop Formation/ResolutionComponents, Helicase/Resolvase Mix, Primosome Complex, DNA PolymeraseIII holoenzyme Complex, Lagging Strand Mix) is made in 1× reactionbuffer.

Then, the five solutions are mixed together in equal volumes to initiatethe reaction. The reaction is incubated for a sufficient time of untilthe reactants are exhausted. Typical incubation times would range from 1hour, 2 hours, 3 hours, 5 hours, 10 hours or overnight (about 16 hours).As stated above, there is no limit to the reaction volume of RPA.Reaction volumes of 25 μl, 50 μl, 100 μl, 1 ml, 10 ml and 100 ml orlarger may be performed in one vessel. Incubation temperature may be atypical laboratory temperature such as 25° C., 30° C., or 37° C.

FIG. 3 shows initiation (FIG. 3A), synthesis (FIG. 3B), and polymeraseamplification (FIG. 3C-3D). First, the primosome loads onto the D-loopformed by RecA nucleoprotein filament invasion (FIG. 3A). The primosomesynthesizes a stretch of RNA primer. Finally, the primosome recruits theclamp loader, which recruits both the sliding clamp dimer and theasymmetric DNA polymerase core (FIG. 3A). Synthesis occurssimultaneously in both the leading and lagging directions. Eventuallylagging strand synthesis stops and the lagging strand clamp is unloaded(FIG. 3B). Synthesis of the leading strand continues until a new site oflagging stand synthesis is formed (FIG. 3B). While leading strandsynthesis continues, a new site of lagging stand synthesis is formed.Lagging strand synthesis continues back to the previous Okazaki fragmentwhere the lagging strand clamp is unloaded (FIG. 3C). DNA Polymerase Iremoves the RNA primer, and fills in the gap while DNA ligase connectsthe two Okazaki fragments forming a continuous lagging strand (FIG. 3D).

Example 4 Establishment of an Amplification Environment Using theAssembly of Heterologous Components E. coli recA(C) and T4 gp32(N)

FIG. 18 shows the results of an experiment in which recA(C) has beencombined with gp32(N) in the presence of pairs of oligonucleotides,Tester3bio (possessing a 5′ biotin label) and Sizer1, Sizer2, Sizer3,Sizer4 or Tester2. These latter unbiotinylated oligonucleotides werepositioned progressively further away from the common Tester3biooligonucleotide. The template was a linear DNA fragment, approximately300 bp, released from a plasmid. Tester3bio was designed to becomplementary to one end of this fragment and included a 5′ overhangrelative to this sequence.

The reaction buffer included Magnesium acetate at 10 mM, required tosupport recA binding to DNA, and 3 mM ATP. Also included was an ATPregeneration system, comprising phosphocreatine and creatine kinase, aswell as dNTPS at 200 μM, and the Klenow fragment of E. coli DNApolymerase I. PEG compound was employed as shown. Double strandedtemplate DNA (0.5 fmoles), derived from a plasmid carrying the E. coliruvB gene, was used as a starting target. The Sizer1, Sizer2, Sizer3,and Sizer4 oligonucleotides did not recognise the other end of thetemplate. Instead, these oligonucleotides were positioned to faceTester3bio with increasing distance between their relative 3′ ends.

After an incubation of 2 hours at 37° C., there was a substantialamplification of specific fragments of the correct size when Tester2, 3,and 4 were used. In the best conditions (with Sizer2), we estimated thatthe amplification product were 10⁴ fold greater than the startingtemplate.

Example 5 The Nature of Amplification Products and the Sensitivity ofthe Reaction Using a Heterologous Assembly of E. coli recA(C) andBacteriophage T4 gp32(N)

FIG. 19 shows the results of an experiment in which recA(C) has beencombined with gp32(N) in the presence of the pair of oligonucleotides,Tester3bio (possessing a 5′ biotin label) and Sizer2, under conditionssimilar to those used in Example 1. PEG compound or PEG 1450 wereemployed as shown and 0.5 fmoles of template was used as a startingtemplate amount. In this example, progressive dilution of the templatewas investigated. Alternatively we explored the use of linearisedstarting template possessing no end that overlaps the primer (by using aClaI digest of the E. coli ruvB gene carrying plasmid), and dilution ofthe Klenow fragment. Amplification of correctly sized fragments occurredin all lanes and was strongest in the case of 0.5 fmoles-startingtemplate in the presence of PEG compound.

When the products of these optimal reactions were electrophoresed onagarose gels and stained with ethidium bromide, a clean band ofdouble-stranded DNA of the correct size was observed. When this samplewas treated with BbvC1 restriction enzyme prior to electrophoresis theexpected increase in gel mobility occurs consistent with a single cut asexpected. Amplification of a product of the correct size was observedwith starting template dilutions of 100-fold, or greater, although theproduct was less abundant and includes a ladder of shorter productsbelow the main band. A similar pattern was observed when uncut templateis employed or when no template is employed. We reasoned that theproteins used in these studies were significantly contaminated with E.coli genomic DNA (naturally carrying the ruvB gene) as they werepurified in single column purifications without the use of nucleases.Consequently we believe this test system generates false positives whenthe sensitivity is high enough.

Example 6 Establishment of an Amplification Environment Using anAssembly of gp32(N) and uvsX(C)

FIG. 24 shows results of an experiment in which uvsX(C) has beencombined with gp32(N) in the presence of the oligonucleotides Tester3bioand Sizer2. The template DNA in this experiment was an EcoRV digestionof the E. coli ruvB gene carrying plasmid used in Examples 1 and 2.Tester3bio recognised one end of an approximately 300 base pair fragmentand included a 5′ overhang relative to the end of the target sequence.Sizer2 recognised the other strand of this template. Thisoligonucleotide was directed toward embedded sequences such that its 3′end was about three and a half helical turns from the end of Tester3bio.

In the presence of PEG 1450, we observed the amplification of theexpected fragment within the 2 hours of the reaction. In the cases whereamplification has occurred, almost the entire of population ofoligonucleotides was consumed indicating an amplification of 3-5×10⁴.The reaction components are indicated on FIG. 24. Included in somesamples were additional components. We found that 200 μM ADP-β-Sincluded in this reaction slightly increased the amount of productformed under these conditions. Conversely, under the conditions usedhere, inclusion of E. coli toposiomerase I was inhibitory to DNAamplification. Under the conditions used, we detected no amplificationwith uvsX(C)delta protein. However, no PEG1450 was included in thesesamples and uvsX(C) also failed to amplify under these conditionswithout PEG1450.

Example 7 Amplification of a Target from Human Genomic DNA Using T4Recombination Proteins

FIG. 30 shows the results of an experiment in which several pairs ofprimers were employed to amplify a specific DNA fragment from humangenomic DNA. The reaction included bacteriophage T4 gp32(C)K3A, uvsX(C)and uvsY(N) proteins, as well as exonuclease deficient Klenow fragment,and proteins comprising the ATP regeneration system to convert ADP andAMP. To detect the specific DNA fragment, we transferred theelectrophoretically separated reaction products to nylon membrane, thenhybridised a biotinylated probe, which recognised a unique non-primerinternal sequence.

Three primer pairs were employed, and in each case a comparison was madebetween no input genomic DNA, 10,000 copies of uncut human genomic DNA,and 10,000 copies of HpaII cut genomic DNA (which generates at least oneend for the primer pairs). In all cases, specific amplification of thedesired DNA sequence occurred, while the efficiency showed variationbetween primer pairs, and between uncut and cut DNAs. In all cases,prior HpaII digestion of the DNA sample was not absolutely required, butimproved the efficiency of amplification. In all cases, input genomicDNA was important. In the best amplification (shown in lane 4), weestimated at least 10¹¹ molecules, indicating an amplification of theapproximate order 10⁷.

Example 8 Sensitivity of lsRPA when Targeting a Complex DNA—HumanGenomic DNA

FIG. 31 shows the results of an experiment in which several pairs ofprimer were employed to amplify a specific DNA fragment from humangenomic DNA. The reaction included bacteriophage T4 gp32(C)K3A, uvsX(C)and uvsY(N) proteins, as well as an exonuclease deficient Klenowfragment, and comprising the ATP regeneration system to convert ADP andAMP. To detect the specific DNA fragment, we transferred theelectrophoretically separated reaction products to nylon membrane. Thenwe hybridised a biotinylated probe, which recognises a unique non-primerinternal sequence.

Three primer pairs were employed and in each case a comparison is madebetween no starting template and approximately 10, 100, 1000, 3000, and10,000 copies of the genomic target. In all cases, clear amplificationwas detected when at least 1000 copies of the genomic target were used(a weak signal is seen with the best primer pair at 100 copies). Weconcluded that during that IsRPA reactions configured in this way werecapable of amplifying DNA from very complex targets with a sensitivityof at least 1000 copies, and potentially higher.

Example 9 Competition Between the Accumulation of Bona Fide Product andPrimer (Template-independent) Artifacts During Reactions

FIG. 32 shows the results of an experiment in which a pair of primerswas employed to amplify a specific DNA fragment from human genomic DNA.Employed in the reaction were bacteriophage T4 gp32(C), uvsX(C) anduvsY(N) proteins, as well as an exonuclease deficient Klenow fragment,and proteins comprising the ATP regeneration system to convert ADP andAMP. PEG 1450 was included at 10% w/v. One of the oligonucleotidesincluded a 5′-biotin so that all reaction products could be observed atthe end of the amplification. Samples were taken at 1, 2 and 3 hours toobserve how the reaction progressed. In one sample, when a minimalamount of uvsY(N) was employed (50 ng/μl), amplification of the correctfragment was observed (see arrow in lane 4). This fragment was cleavedby BstXI to the expected size fragment, indicating it was principallydouble-stranded. However the fragment was less abundant than apparentlytemplate-independent bands that also accumulated during the reaction.The size and template-independent nature of these bands suggested thatthey were primer artifacts, e.g., primer dimers and/or snapbacksynthesis products. The absence of amplification of the specificfragment suggested that, at uvsY(N) concentrations greater than 50ng/μl, the reaction occurred suboptimally. This was borne out by laterexperiments.

Example 10 Optimisation of Reaction Composition to Severely Limit orEliminate Primer Artifacts and Enable Sensitive Noise-free Amplificationfrom Complex Templates

FIG. 36 shows the results of an experiment in which a pair of primerswas employed to amplify a specific DNA fragment from human genomic DNA.Employed in the reaction were bacteriophage T4 gp32(C), uvsX(C) anduvsY(N) proteins, as well as an exonuclease deficient Klenow fragment,or Bst polymerase, and proteins comprising the ATP regeneration systemto convert ADP and AMP. One of the oligonucleotides included a 5′-biotinso that all reaction products could be observed at the end of theamplification. Amplified fragments were visualised following separationof fragments by size by running a small sample of the reaction on anacrylamide gel. In this experiment, uncut human genomic DNA was titratedfrom zero copies, 45 copies, and then doublings in target copy number upto 2880. Slightly different conditions were employed in this experimentfor each of the two polymerase species with regard to both buffer andtemperature. The reaction with the Klenow fragment was performed at 37°C., while that with Bst polymerase was performed at 42° C. The detailsof the buffer composition are given in the figure description.

Of note, and important to the efficiency of reactions under theseoptimised conditions, PEG compound was included at 5% final weight tovolume in both cases. Both polymerases have effectively amplified thecorrect fragment, and in some cases, utilised most of the availableprimers. Under the conditions used for the Klenow fragment, thesensitivity was so great that a weak signal was observed even in thezero copies lane presumably reflecting contamination with a quantity ofhuman DNA representing less than the 45 copies present in the laneimmediately adjacent. At the level of sensitivity that is demonstratedhere, it was difficult to eliminate trace levels of contamination fromthe equipment that was used leading to signals in the negative controls.Routine employment of conditions similar to those utilised in theKlenow-mediated amplification proved effective for noise-freeamplification of numerous primer pairs in later experiments. Thissuggested that these conditions were close to one optimum for reactionsinvolving this set of protein components.

Example 11 Experimental Methods for Production of Clones and Proteins

All clones have been constructed by cloning PCR amplified products fromE. coli, T4 phage, B. subtilis, or Phi-29 phage. All stock organismsused for amplification were obtained from a public source at the DSMZ.Cloned DNA's used for protein expression have in general been clonedinto pET vectors (e.g., pET-21) with the insertion of a hexahistidinepeptide tag at either the N or C terminus during the PCR amplificationof the fragment, or into pQE vectors (e.g., pQE31) in the case of Pol Ifrom B. subtilis (Bsu polymerase). In this disclosure all proteinscontaining an N terminal tag are referred to as the protein namefollowed by (N), e.g. gp32(N), or if containing the tag at the Cterminus the name is followed by (C), e.g. gp32(C). Additionally we haveconstructed several clones to produce otherwise modified proteins. Theseinclude a recA(C) with a deletion of the last 17 amino acid residues ofthe native protein, referred to as recA(C)delta17. A similar form of theT4 UvsX(C) protein has been generated and is referred to as UvsX(C)delta21. We have also constructed mutant forms of gp32, which modify eitherlysine 3 or arginine 4.

All proteins were overexpressed in E. coli and purified usingconventional protocols. Proteins have generally been purified bystandard procedures on Nickel resin in 1 M NaCl and phosphate buffer.Proteins were eluted with 250 mM imidazole and dialysed into appropriatebuffers. Proteins produced from clones generated in-house include: E.coli recA(C), E. coli SSB(N), E. coli PriA(N), E. coli PriB, E. coliPriC, E. coli DnaB, E. coli DnaC, E. coli DnaC810, E. coli DnaT, E. coliRuvA, E. coli RuvB, T4 phage UvsX(C), T4 phage UvsX(N), T4 gp32(N), T4gp32(C), T4 gp32(C)K3A, T4 phage gp32(C)R4Q, T4 phage gp32(C)R4T, T4phage gp32, T4 phage gp32 K3A, T4 phage gp32R4Q, T4 phage gp32R4T, T4phage UvsY(N), T4 phage UvsY(C), T4 phage gp43, T4 phage gp43(exo-), E.coli Klenow fragment, E. coli Klenow exo-. Untagged gp32 proteins werepurified by a 2-column procedure involving DEAE sepharose anionicexchange followed by binding to single-stranded DNA cellulose matrix.

DNAs Used in RPA Reactions.

We have employed several different target DNAs in this study, and anumber of oligonucleotides. The sequence of the relevant section of thetemplates, and the sequence of the oligonucleotides is given below.

The E. coli RuvB Gene Target

The sequence of the EcoRV fragment of the RuvB gene is given below.

(SEQ ID NO: 1) ATCATGATTGGTGAAGGTCCGGCGGCACGCTCCATTAAAATTGATTTGCCGCCGTTTACCCTGATTGGTGCAACCACGCGCGCAGGTTCGCTGACATCACCGTTGCGCGACCGTTTTGGTATTGTGCAACGTCTGGAGTTTTATCAGGTGCCGGATCTGCAATATATCGTCAGTCGCAGCGCACGCTTTATGGGGCTTGAGATGAGTGATGACGGCGCGCTGGAAGTTGCTCGTCGCGCTCGCGGTACGCCGCGCATTGCCAACCGTCTGCTGCGTCGAGTGCGTGATTTCGCCGAAGTGAAGCACGATGGCACCATCTCGGCAGAT

The sequence of oligonucleotides targeting this template mentioned inthis study are given below:

Tester2 (SEQ ID NO: 2) CTAGCGATGGTGCCATCGTACAGAATTCCCTCAGCATCTGCCGATester3 (SEQ ID NO: 3) CTCACTATACCTCAGCATCATGATTGGTGAAGGTCCGGCGGCACTester1 bio (SEQ ID NO: 4)5′-biotin-GCTAATACGACTGACTATACCTCAGCATCATGATTGGTGA AGGTCCGGCGGCAC Tester3 bio (SEQ ID NO: 5)5′-biotin-CTCACTATACCTCAGCATCATGATTGGTGAAGGTCCGGCG GCAC Sizer1(SEQ ID: 6) CTATGCGAATTCAGCGAACCTGCGCGCGTGGTTGCACCAATCAGGG Sizer2(SEQ ID NO: 7) CTATGCGAATTCGGTGATGTCAGCGAACCTGCGCGCGTGGTTGCA Sizer3(SEQ ID NO: 8) CTATGCGAATTCTCCAGACGTTGCACAATACCAAAACGGTCGCGC Sizer4(SEQ ID NO: 9) CTATGCGAATTCCGTGCGCTGCGACTGACGATATATTGCAGATCC Gen2bio(SEQ ID NO: 10) 5′-biotin-ATCTGCCGAGATGGTGCC

The sequence of part of the human angiotensin converting enzyme targetedin this study is shown below:

(SEQ ID NO: 11) AACCAACTCCGCCCCGGGCCACGGCCTCGCTCTGCTCCAGGTACTTTGTCAGCTTCATCATCCAGTTCCAGTTCCACGAGGCACTGTGCCAGGCAGCTGGCCACACGGGCCCCCTGCACAAGTGTGACATCTACCAGTCCAAGGAGGCCG GGCAGCGC

Underlined are HpaII restriction sites that have been targeted withHpaII in the preparation of some DNAs in some experiments.

The sequence of oligonucleotides used to target part of the human ACEgene are shown below:

Up3 (SEQ ID NO: 12) ATTCGTCAGCCTCGCTCTGCTCCAGGTACTTTGTCAGCTTCATC Down1(SEQ ID NO: 13) GCCTCCTTGGACTGGTAGATGTCACACTTGTGC Down2 (SEQ ID NO: 14)GCGCTGCCCGGCCTCCTTGGACTGGTAGATGTCACACTTGTGC Down3 (SEQ ID NO: 15)TATGCGAATTGCCTCCTTGGACTGGTAGATGTCACACTTGTGC Angio1bio (SEQ ID NO: 16)5′-biotin-GCCTCCTTGGACTGGTAGATGTCACACTTGTG Angio3 (SEQ ID NO: 17)GGCCACGGCCTCGCTCTGCTCCAGGTACTTTGTCAGCTTCATC

Example 12 Experimental Results and Analysis

FIG. 9 shows the results from investigations into the nature ofdouble-stranded DNA targets and targeting oligonucleotides. Experimentsusing either supercoiled templates or linearised DNAs suggested thatrecA catalyses the formation of intermediates capable of supportingpolymerase elongation most readily on supercoiled DNA, or the ends oflinearised DNA. Shown are the results of an experiment in which thebiotinylated oligonucleotide, Tester3bio, has been incubated with eithersupercoiled target DNA, or a target template linearized with EcoRV, orClaI. This generated an end that overlapped with the oligonucleotide orembedded sequences respectively. The reaction solution included 20 mMTris-acetate pH 7.9, 10 mM Mg-acetate, 13 μg rec A, 1 μg E. coli SSB, 27mM phosphocreatine, 1 U creatine kinase, 0.2 μM Tester3bio, 3 mM ATP,200 μM dG, dC, and dT; 1 mM dA, 50 U Klenow, 0.5 pmoles template, 120 ngrecO, 120 ng recR, 0.5 μM dnaB, and 0.5 μM dnaC810. E. coli recO andrecR proteins, as well as dnaB and dnaC810 proteins, were included inthis experiment although they did not significantly affect the results.After 2 hours of reaction at 37° C., the reaction was precipitated andrun on a 6% denaturing gel, transferred to nylon membrane, and incubatedwith streptavidin-HRP prior to performing ECL to detect reactivematerial. In each reaction, 0.5 pmoles of template was used. Included onthe gel as a control for size and amount was 0.5 pmoles of biotinylatedPCR fragment (labeled CON). Other reaction components and conditions areindicated on the figure.

FIG. 10 shows backfire synthesis. Backfire synthesis occurs when arecombinase-coated targeting oligonucleotide possessing a 5′ overhanginvades a duplex DNA end in the presence of a suitable polymerase anddNTPs. This new duplex region is stable to subsequent branch migrationand can be utilised as a platform for other applications. Forward fireis the elongation of the invading oligonucleotide, which also occurs inthese reactions. Shown are the results of experiments to detect theactivity of polymerases on intermediates formed when the oligonucleotideTester3, possessing a 5′ overhang relative to the end of a linearizedtarget DNA, is incubated with various templates.

In part A, the template used is a double-stranded PCR product generatedsuch that the product has a biotin label at the 5′ end of the strandcomplementary to the targeting oligonucleotide. This fragment isotherwise similar to the EcoRV fragment released from a plasmid carryingthe E. coli RuvB gene used elsewhere in this study, and which is atarget for the Tester3bio oligonucleotide. The reaction solutionincluded 10 mM Mg-acetate, 7.5 μg recA, 1 μg SSB, 27 mM phosphocreatine,1 U creatine kinase, 0.3 μM Tester3bio, 3 mM ATP, 200 μM dNTPs, 50 UKlenow, 0.5 pmoles biotinylated template. Optionally, we included 0.5 μMruvA and 0.5 μM ruvB; or 1 μM ruvA and 1 μM ruvB; or 1.5 μM ruvA and 1.5μM ruvB. The final volume was 30 μl. Incubation was carried out for 1hour at 37° C. In the presence of recA, the biotinylated strand of thetarget was extended by 16 bases, as would be expected if a recombinationintermediate were accessible by a polymerase to copy the overhang regionof the invading oligonucleotide.

In part B, the reaction is configured in a similar manner except thatthe template is not biotinylated, and the invading oligonucleotide isbiotinylated. Several polymerases were investigated in this experiment,and only unmodified Klenow fragment gave a significant production ofproduct. In this experiment, we also investigated including a smalloligonucleotide designed to recognise the target directly downstream ofthe Tester3 targeting site. The reaction solution included 10 mMMg-acetate, 10 μg recA, 1 μg SSB, 27 mM phosphocreatine, 1 U creatinekinase, 0.3 μM Tester3bio, 3 mM ATP, 200 μM dNTPs, 50 U Klenow, 0.5pmoles unbiotinylated template. Optionally, we included 5 μl preloadedstable ATPγS oligonucleotide. The final volume was 30 μl. Incubation wascarried out for 1 hour at 37° C. We pre-incubated with recA in thepresence of ATP-γ-S in an effort to load recombinase stably onto it.Pre-load solution included 10 mM Mg-acetate, 2.5 μg recA, 50 μM ATPγS,and 0.15 μM oligonucleotide. The pre-load solution was added into theTester3bio invasion/extension mixture. In all cases, the yield ofproduct was decreased by inclusion of this premixed material. Based onour data, we believe that the presence of ATP-γ-S (final concentration˜8 μM) in the reaction was mildly inhibitory. The purpose of thisexperiment was to address whether the presence of a stable 3-strandedhybrid formed immediately downstream of the Tester3 targeting site wouldstabilise these invasions to branch migration.

FIG. 11 shows uses of backfire synthesis. Backfire synthesis can beuseful because it generates a branch migration resistant platform thatcan be employed in applications other than straightforward forward fire.Some examples are shown here, including introduction of a nicking enzymetarget site, introduction of an RNA polymerase promoter, and the lineargeneration of short dsDNA fragments through successiveinvasion/synthesis/cleavage events. If a restriction enzyme site isincluded in the additional overhang sequence such that after targeting asuitable linearized fragment, backfire synthesis will generate theduplex target for the restriction enzyme.

The enzyme can then cut the sequence releasing a short double-strandedDNA, and a longer double-stranded DNA, which is a target for furtherinvasion events.

In FIG. 11B, the 5′ overhang of a targeting oligonucleotide is designedsuch that should backfire synthesis occur, a target for a nickingendonuclease is generated. In the presence of the nicking endonuclease,for example BbvC1a or b, a suitable polymerase, for example the Klenowfragment, can extend from the nick and displace a DNA strand. Multiplestrands may be run-off by successive nicking and elongation from asingle template. In FIG. 11C, the 5′ overhand that is converted toduplex by backfire synthesis contains the sequence of an RNA polymerasepromoter, such as the phage T7 RNA polymerase gene. In the presence ofthe necessary polymerase and suitable nucleoside triphosphates,transcription can initiate downstream of the promoter to generate an RNAas shown. The presence of a break in the non-template strand is notpredicted to prevent successful elongation. RNA products might be usedin some form of amplification reaction, or for other purposes.

FIG. 12 shows that single stranded binding proteins facilitaterecombinase invasion and primer extension. Both E. coli SSB andbacteriophage T4 gp32 with an N-terminal His tag (gp32(N)) are able tostimulate recA-mediated invasion/elongation on a linear DNA template.The results of an experiment are shown in which 0.5 pmoles of targettemplate (the EcoRV fragment released from a plasmid carrying the E.coli ruvB gene) was incubated with the Tester3bio oligonucleotide thatoverlaps one end of the template. Either the E. coli SSB protein, or theT4 gp32(N) protein was included to stimulate the reaction. The reactionsolution included 10 mM Mg-acetate, 6 μg rec A, 8.8 μg gp32 or 1 μg SSB,27 mM phosphocreatine, 1 U creatine kinase, 0.3 mM Tester3bio, 3 mM ATP,200 μM dNTPs, 50 U Klenow, 0.5 pmoles template.

Optionally, we included 120 ng recO and 120 ng recR. The final volumewas 30 μl. Incubation was carried out for 1 hour at 37° C. Otherreaction components and conditions are indicated in the figure. Thefigure also shows the general relationship of the primer and target DNA.In the reactions where E. coli recO and recR proteins were included,little effect was seen from their addition under these conditions.Invasion and elongation appeared to have proceeded in all cases, and thegp32(N) appeared to have stimulated synthesis even better than E. coliSSB, although it was used at higher concentration in this experiment.

FIG. 13 shows the requirement for a minimal oligonucleotide length oroverhang for invasion and elongation during end targeting of lineartemplates. The results of an experiment are shown in which 0.5 pmoles oftarget template (the EcoRV fragment released from a plasmid carrying theE. coli ruvB gene) was incubated with the either the Tester3biooligonucleotide. This oligonucleotide overlaps one end of the template,or the Gen2bio oligonucleotide, which is flush to the other end of thetemplate and is only 18 residues long. The reaction solution included 10mM Mg-acetate, 27 mM phosphocreatine, 1 U creatine kinase, 0.2 μMTester3bio or Gen2bio, 10 mM dATP, 3 mM ATP, 200 μM dNTP mixture, 50 UKlenow or Phi29 polymerase, 13 μg recA(C), 1 μg E. coli SSB, and 0.5pmoles template. The final volume was 30 μl. Incubation was carried outfor 2 hours at 37° C., and 2 μl or the reaction was loaded in each laneof the gel. Other reaction components, conditions, and the generalrelationship of the primers and target DNA are indicated on the figure.Invasion and elongation appeared to have proceeded efficiently in thepresence of the Klenow fragment, less efficiently with the Phi29polymerase, and less well with the Gen2bio primer and the Klenowfragment. We concluded that a minimal primer length and/or an overhandrelative to the template was-required to stimulate efficient invasionand elongation.

FIG. 14 shows paranemic (A-E) and pletonemic (F-H) joints. For paranemicjoints, the interaction of the recombinase filament with DNA stimulatesunwinding (FIG. 14A). The unwound region moves with the homology search(FIG. 14B). The homology is found (FIG. 14C). The recombinasedissociates and a new duplex attempts to rewind (FIG. 14D). Because ittopologically restrained, the ‘outgoing’ strand is forced to rewindaround the new duplex (FIG. 14E). This state is highly unfavorable andunstable, and cannot always be managed by SSBs (FIG. 14E). Forplectonemic joints, the interaction of the recombinase filament with DNAstimulates unwinding (FIG. 14F). If strand exchange overlaps with a DNAend, the ‘outgoing’ strand is freed and can relax as the incoming oligoand its complement rewind as the recombinase dissociates (FIG. 14G).This forms a deconstrained product, and a single-strand DNA bindingprotein inhibits branch migration (FIG. 14H).

This figure compares the likely events that occur when a nucleoproteinfilament initiates strand exchange with a homologous sequence located atthe end of a linearized duplex (right side of figure), or within aduplex which lacks homology on either side (left side of figure).Starting with the left side, once the nucleoprotein filament has locatedthe correct sequence it will pair the searching DNA to its complement,and one strand of the original duplex becomes unpaired. In fact, theexchange complex consists of 3 strands, which are relatively under-woundand stabilised by the recombinase. As the recombinase begins todisassemble in a 5′ to 3′ direction, the under-wound 3-strandedintermediate becomes unstable. For the new duplex to regain the normalconformation of relaxed DNA, it must rotate. However, in doing so, itmust co-rotate the outgoing strand, as it is linked upstream anddownstream to its original partner. This results in over-winding theoutgoing strand, as it has to make the same number of turns but take alonger path around the new duplex, and is energetically unfavourable.Consequently there is a requirement for single-stranded binding proteinswith very stable DNA interactions to permit such structures to exist forany significant time. Alternatively the right side of the diagramindicates that should the exchange include an end of the duplex thenexchange can cause the complete release of the outgoing strand at oneend and thus permit it to rotate freely unconstrained by the otherstrands involved in recombination. This leads to a stable situation inwhich the new duplex is free to rewind after recombinase disassembly,and single-stranded DNA binding proteins need only deter spontaneousbranch migration.

FIG. 15 shows the effect of crowding agents. In the presence ofpolyethylene glycols, gp32(N) and recA recombinase can mediate multipleinvasion events on single templates without a requirement forregeneration of the template ends that would permit 5′ overhangs in thetargeting oligonucleotide. FIG. 15A shows the results of an experimentin which either the Tester1bio, or Tester3bio oligonucleotides (whichdiffer in the length of 5′ overhang relative to the template) wereincubated with the EcoRV fragment released from a plasmid carrying theE. coli ruvB gene, or the ClaI digest of the plasmid, in the presence orabsence of 10% PEG 8000. The reaction solution included 10 mMMg-acetate, 10.6 μg recA, 8.8 μg gp32, 27 mM phosphocreatine, 1 Ucreatine kinase, 0.3 mM Tester3bio or Tester1bio, 3 mM ATP, 200 μM dNTPmixture, 50 U Klenow, and 0.5 pmoles template (species indicated infigure). Optionally, we included 120 ng recO and 120 ng recR. PEG8000was included as shown. The final volume was 30 μl. Incubation wascarried out for 1 hour at 37° C.

The diagram shown represents the relationship of the oligonucleotides tothe two possible templates. In particular, both oligonucleotidesrecognised an embedded sequence within the ClaI fragment. In each case,0.5 pmoles of template was used, other conditions were carried out asindicated. Both primers stimulated invasion/elongation on the EcoRVtemplate. Based on signal intensity, approximately one elongationoccurred per target template. However, in the presence of 10% PEG 8000,the intensity of the fully elongated fragment was significantly greaterthan in its absence and stronger than the 0.5 pmoles of controlbiotinylated PCR product. The strongest signal was seen with theTester3bio oligonucleotide. In that case, we estimated at least 10invasion/run-ons occurred per template.

In FIG. 15B, we compared the stimulation of invasion/elongation in 10%w/v of various commercially available polyethylene glycols. The reactionsolution included 10 mM Mg-acetate, 10.6 μg recA, 8.8 μg gp32, 27 mMphosphocreatine, 1 U creatine kinase, 0.3 mM Tester3bio or Tester1bio, 3mM ATP, 200 μM dNTPs, 50 U Klenow, and 0.5 pmoles RV template. PEGspecies were included as shown. There was significant variation observedin the degree of stimulation. PEG compound (MW=15,000 to 20,000)appeared to be the most effective, followed by PEG1450.

FIG. 16 shows the effect of end targeted amplification using leadingstrand RPA. Amplification comprising several rounds of invasion andextension was demonstrated, achieving at least a 10 fold amplificationfrom 0.05 pmoles of template. In this experiment, we have employed pairsof oligonucleotide primers to establish an amplification reaction. Shownschematically is the relationship of the oligonucleotides used to theEcoRV fragment from a plasmid carrying the E. coli ruvB gene, which wasused as template. The reaction solution included 10 mM Mg-acetate, 6 μgrecA, 8.8 μg gp32(N), 27 mM phosphocreatine, 1 U creatine kinase, 0.3 mMTester3bio, 0.3 mM variable oligonucleotide, 3 mM ATP, 200 μM dNTPs, 10%PEG compound, 50 U Klenow, and 0.5 pmoles template. Additional proteinswere used as indicated.

Tester3bio included a 16-nucleotide overhang relative to the startingtemplate, while Tester2 included a 21-nucleotide overhang and wastargeted to the other end of the template. Phospho1 was used as anoligonucleotide with a phosphorothiorate backbone. This oligonucleotidewas 15 residues long, and was flush to the target end. Phospho-1 waspredicted not to interact with recombinase or single-stranded DNAbinding protein as it lacked a phosphate backbone. However, it waspredicted to function in straightforward solution hybridisation. Acontrol fragment of biotinylated PCR product was employed to demonstratethe signal intensity of 0.5 pmoles of DNA, and was also the precise sizeof the starting template. The reaction products were run on a 6%denaturing gel, transferred to nylon, and bound with streptavidin-HRPprior to performing enhanced chemiluminescence to reveal thebiotinylated products of the reactions.

In all cases, successful invasion and elongation with the biotinylatedTester3bio has occurred as seen by presence of fully elongated products.The products were slightly slower mobility that the control, due to thepresence of overhangs on the oligonucleotides. Furthermore, there wasevidence for several rounds of invasion/run-ons as the signal intensitywas at least as great as the 0.5 pmoles control (we initiated thereaction with only 0.05 pmoles). There was a significant accumulation ofa product roughly 37 nucleotides larger than the control. This waspredicted to arise from Tester3bio elongating on a strand previouslycopied from, and including the overhang from, the opposing primer. Twoexposures of the same gel are shown.

The inclusion of various different proteins, which are normally involvedin DNA metabolism, had varying effects. DNA gyrase, and toposiomerase I(human) decreased the yield of amplification product, and thetopoisomerase profoundly reduced the generation of shorter elongationproducts. Inclusion of E. coli ruvA and ruvB also lead to a generalreduction in product formation. E. coli priA increased the amount ofproduct formed, and significantly increased the number of shorterproducts formed. Inclusion of E. coli dnaB and dnaC810 protein slightlyincreased the amount of product formed. Note that a significantlystronger signal detected in reactions containing Phospho-1oligonucleotide in comparison to Tester3bio alone. This suggested thatPhospho-1 was able to hybridise with displaced strands and lead toformation of duplex DNA.

FIG. 17 shows leading strand RPA and Klenow processivity. In thisexperiment, we have employed pairs of oligonucleotide primers in aneffort to establish an amplification reaction in a manner similar tothat shown in FIG. 16, except using a further 100-fold dilution of thestart template. The reaction solution included 10 mM Mg-acetate, 6 or 12μg recA, 8.8 or 14.3 μg gp32, 27 mM phosphocreatine, 1 U creatinekinase, 0.3 or 0.9 μM Tester3bio, 0.3 or 0.9 μM Tester2, 3 mM ATP, 200μM dNTPs, 10% PEG compound, 50 U Klenow, and 0.5 pmoles template. Thefinal volume was 30 μl. Incubation was carried out for 2 hours at 37° C.Shown schematically is the relationship of the oligonucleotides used toan EcoRV fragment from a plasmid carrying the E. coli ruvB gene, whichis used as target template. Tester3bio included a 16-nucleotide overhangrelative to the starting template, while Tester2 included a21-nucleotide overhang, is targeted to the other end of the template,and encodes an EcoRI site within the overhang. A control fragment ofbiotinylated PCR product was employed to demonstrate the signalintensity of 0.5 pmoles of DNA, and was also the precise size of thestarting template. The reaction products were run on a 6% denaturinggel, transferred to nylon, and bound with streptavidin-HRP prior toperforming enhanced chemiluminescence to reveal the biotinylatedproducts of the reactions.

The concentration of the oligonucleotides, gp32(N) and the recA(C) werevaried, and we have also investigated whether the inclusion of EcoRIrestriction enzyme, which can cleave part of the additional sequenceincorporated by the Tester2 overhang, has any effect on the reaction. Inmost cases, there was evidence of some limited degree of amplificationof the expected size of fragment, but there was principally generationof shorter DNA fragments. We deduced that the relatively pooraccumulation of bona fide full length product may occur at these moredilute template concentrations because the poor processivity of the E.coli DNA polymerase I Klenow fragment (10-50 nucleotides) results inmost interactions generating short fragments, which is more significantat low target template concentrations.

FIG. 18 shows spacing dependence of RPA primers. As a consequence ofearlier results, we attempted to establish whether decreasing thedistance between primer pairs would result in an increase inamplification efficiency. To test this, we employed a series ofoligonucleotides, Sizer1, 2, 3, and 4, which were positioned atincreasing distances away from the 3′ end of the Tester3biooligonucleotide. All Sizer oligonucleotides included the EcoRI overhangindicated in the bottom right side of the figure. The sequence of thetarget DNA, an EcoRV fragment from a plasmid carrying the E. coli ruvBgene, and the position of the oligonucleotides used are shown. Thereaction solution included 10 mM Mg-acetate, 6 μg recA, 8.8 μg gp32, 27mM phosphocreatine, 1 U creatine kinase, 0.3 μM Tester3bio, 0.3 μMvariable oligonucleotide, 3 mM ATP, 200 μM dNTPs, 10% PEG compound, 50 UKlenow, 5 U EcoRI, and 0.5 pmoles template. The final volume was 30 μl.The reaction solution was incubated for 2 hours at 37° C. Otherreactions conditions are indicated on the figure.

A control fragment of biotinylated PCR product was employed todemonstrate the signal intensity of 0.5 pmoles of DNA, and was also theprecise size of the starting template. The reaction products were run ona 6% denaturing gel, transferred to nylon, and bound withstreptavidin-HRP prior to performing enhanced chemiluminescence toreveal the biotinylated products of the reactions. Specific fragments ofthe expected lengths were efficiently amplified from 0.5 fmoles ofstarting template when Sizer2, 3, and 4 were employed. The yield ofproduct decreased somewhat however as the length of the productincreased. Little or no product of the expected size is produced whenSizer1 was used. Lane 4 was estimated to contain ˜4×10⁴ foldamplification. This primer included the shortest inter-oligonucleotidedistance of only 25 nucleotides. This suggested there is a minimalrequired distance to separate the ends of the oligonucleotides, althoughother explanations such as poor primer behaviour could also explain theresult. Included in the experiment were several samples containing notemplate DNA. In the case of Sizer2, a faint band of approximately theexpected size is observed even in the absence of exogenous DNA. Based ona variety of data we believe that this arises from contamination of ourprotein preparations with significant quantities of E. coli genomic DNA.

FIG. 19 shows that RPA products are largely double stranded. RPAreaction can generate double-stranded DNA products as evidenced byagarose gel electrophoresis and restriction enzyme cleavage. However,under the conditions used here, there were significant decreases inreaction efficiency if the starting template dropped significantly below0.5 fmoles. Furthermore, signals observed in the water-only controlsuggested significant genomic contamination of E. coli DNA. In thisexperiment we have incubated 0.5 fmoles, or more dilute quantities, ofthe fragment detailed in FIG. 10 with Tester3bio and Sizer2 under theconditions indicated. The reaction solution included 10 mM Mg-acetate, 6μg recA, 8.8 μg gp32, 27 mM phosphocreatine, 1 U creatine kinase, 0.3 μMTester3bio, 0.3 μM Sizer2, 3 mM ATP, 200 μM dNTPs, 10% PEG compound, 50U Klenow, and 0.5 pmoles template or dilution indicated in figure. Thefinal volume was 30 μl.

We have included a no DNA control, progressive dilution of the template,and investigated initiating the reaction on embedded template (the ClaIfragment detailed on FIGS. 1 and 7), of using PEG 1450, and diluting theKlenow fragment. A fraction ( 1/10) of the reaction products were run ona 6% denaturing gel, transferred to nylon, and bound withstreptavidin-HRP prior to performing enhanced chemiluminescence toreveal the biotinylated products of the reactions (FIG. 19A). A furtherfraction ( 3/10) was phenol extracted, precipitated, and run on a 2%agarose gel and stained with ethidium bromide (FIG. 19B). A finalfraction ( 3/10) was cut with BbvC1 and electrophoresed on a 2% agarosegel alongside equivalent uncut DNA (FIG. 19C).

Lane 2 (FIG. 19A) was estimated to contain 5×10⁴ fold amplification,which corresponded to 10¹³ molecules of final product. In the presenceof 0.5 fmoles of starting template, an extremely robust amplification ofthe expected size fragment was seen as evidenced by denaturing gelelectrophoresis. Furthermore, when part of the sample waselectrophoresed on agarose a strong clean band of precisely the correctsize for a double-stranded DNA product was observed. This product couldbe cut by BbvC1 to yield a slightly smaller fragment of the expectedsize. Dilution of the template by 100-fold or more resulted in asignificantly less intense band, and a much larger quantity of fragmentsshorter than the expected length. This was determined by denaturing gelelectrophoresis and by agarose gel electrophoresis. A similar patternwas observed when ClaI cut DNA was used, or if no DNA was used. Webelieve that when less than a threshold quantity of DNA is used underthese conditions, there is a suboptimal amplification reaction, whichleads to heterogeneous products, and furthermore that our samples arehighly contaminated with E. coli genomic DNA from the single-steppurification procedures used in generating our recombinant proteins usedhere.

FIG. 20 shows activity of a recA C-terminal truncation mutant. RecAproteins with a deletion of the C-terminal acidic peptide (recA(C)A) areactive in promoting strand exchange and extension in a linear templaterun-on assay. However, there was some suggestion that the optimalprotein concentration was lower than that with the recA(C) protein. Thisexperiment addresses whether a C terminal truncated form of the E. colirecA protein, described elsewhere, could be used successfully ininvasion/extension reactions. Shown is the result of a single-sidedrun-on assay using either the E. coli recA(C) protein or the E. colirecAA17(C) protein, which lacks the 17° C.-terminal acidic residues. Therelationship between primers and template and other experimentalconditions are indicated. The reaction solution included 10 mMMg-acetate, 27 mM phosphocreatine, I U creatine kinase, 0.3 μMTester3bio, 3 mM ATP, 200 μM dNTPs, 50 U Klenow, 0.5 pmoles RV template,and recA species and amount as indicated in figure. PEG was included asshown. The final volume was 30 μl. The solution was incubated for 2hours at 37° C. Reactions were performed with or without 10% PEG 1450,and with the indicated quantities of the respective recombinase. Bothrecombinases successfully supported invasion/extension, although underthe conditions used here there appears to be a different optimum for theamount of protein required.

FIG. 21 shows Modified gp32 proteins. Shown is a schematicrepresentation of T4 gp32 proteins used in this study and the positionof various modification and mutations.

FIG. 22 shows activity of gp32 proteins. Significant variations confirmthat gp32 cooperativity has a substantial effect on the rate ofinvasion/extension reactions, and further confirms that gp32(N) displaysa significant decrease in cooperativity consistent with interferencewith the function of the N-terminal B domain. Shown are the results ofan experiment in which linear run-ons were generated using as templatethe EcoRV fragment of a plasmid carrying the E. coli RuvB gene and theTester3bio oligonucleotide. The reaction solution included 10 mMMg-acetate, 27 mM phosphocreatine, 100 ng/μl creatine kinase, 400 nMTester3bio, 3 mM ATP, 200 μM dNTPs, 10 U chicken myokinase, 8 μg C-taguvsX, 7.5 or 15 μg gp32 (each species), 50 U Klenow, and 0.5 pmolestemplate; no PEG was included. The final volume was 30 μl. The solutionwas incubated for 2 hours at 37° C. Reactions contained uvsX(C) andvarious gp32 forms as indicated. Two concentrations of each gp32 formwere used in this experiment. To analyze the reaction products, 2 μl ofthe total volume (30 μl) was loaded onto the gel.

In all cases, elongated products of the oligonucleotide were generatedthat extend up to a length consistent with full length run-ons. We notethat in this experiment there was a gel artifact which we occasionallyobserved. We observed a slower mobility shadow of the bands. This wasseen in the PCR control fragment lane, also. The smallest quantity ofproduct was formed when using the gp32(C) protein, which was consistentwith it permitting only a low level of recombinase-loaded filaments tobe present in the reaction. A smaller quantity of product was formedwith 15 μg compared with 7.5 μg, consistent with the notion that higherconcentrations decreased the efficiency of recombinase loading.

The gp32(C)K3A protein was predicted to be the next most cooperativeform. Consistent with this, it produced a limited number of full-lengthproducts when 15 μg of protein is used.

However, the number of run-ons was greater than that observed witheither quantity of gp32(C), indicating that there were morerecombinase-loaded filaments in the reaction and a higher rate ofinvasion/elongation. When 7.5 mg of gp32(C)K3A was used, there was adramatic change in the quantity of product formed. One explanation isthat an increased rate of invasion/elongation in this reaction couldlead to the out-titration of the gp32(C)K3A by single-stranded DNArun-offs. Under these conditions, most of the oligonucleotide wouldbecome coated with uvsX(C), leading to a high invasion rate and to aninability to stabilise the outgoing strand and coat it with gp32. Thiswould result in shorter truncated products, some of which would befolded back on themselves, self-primed, and formed into a variety ofother such products. This suggested that the rate of invasion/elongationof gp32(C)K3A was notably higher for this protein than gp32(C).

By comparing the intensities of the products produced when using 15 μgof each protein, we estimated that reactions containing gp32(C)K3A havean invasion/elongation rate of approximately 10 times that of gp32(C).All of the other gp32 proteins tested in this experiment produced apattern similar to that seen when gp32(C)K3A was employed and largeamounts of product. This was the case even when 15 μg of the relevantprotein was employed, suggesting that they all exhibited lowercooperativity than either gp32(C) or gp32(C)K3A. Notably, however, bothgp32(N) and gp32(C)R4T produced significantly less product when only 7.5μg of protein was used when compared with 15 μg. This contrasted to thesituation with the other proteins. On this basis, we suggest thatgp32(N) and gp32(C)R4T possess a similar degree of cooperativity. Anearlier study has suggested that gp32K3A and gp32R4Q are of similarcooperativity. However, our data would suggest that gp32(C)R4Q liessomewhere between gp32(C)K3A and gp32(C)R4T with regard to its behaviourin supporting invasion/synthesis.

FIG. 23 shows invasion and extension using uvsX. This experimentaddresses whether a C terminal truncated form of the bacteriophage T4uvsX protein could be used successfully in invasion/extension reactions.Shown are the results of single-sided run-on assays using either theuvsX(C) protein or the uvsX.DELTA.21(C) protein which lacks the 21°C.-terminal acidic residues. The reaction solution included 10 mMMg-acetate, 27 mM phosphocreatine, 1 U creatine kinase, 0.4 μMTester3bio, 3 mM ATP, 200 μM dNTPs, 50 U Klenow, 1 U chicken myokinase,uvsX(C) or uvsXA21(C), 8.8 μg gp32(N), and 0.5 pmoles RV template. Thefinal volume was 30 μl. The solution was incubated for 2 hours at 37°C., and 2 μl or reaction mixture was loaded onto each lane of the gel.The relationship of the template and oligonucleotides and otherexperimental conditions are indicated in the figure. Reactions wereperformed with the indicated quantities of the respective recombinase.

Both recombinases successfully supported invasion/extension. However,under the conditions used here, there appears to be a different optimumfor the amount of protein required. In the case of uvsX(C), the rate ofinvasion/extension increased progressively with the concentration ofprotein within the range tested. However for the uvsXA21(C) protein, therate was inhibited at higher concentration and the overall level ofproduct production was lower under these conditions. In contrast torecA-mediated invasion/extension in similar reactions, uvsX(C), appearedto stimulate multiple invasion/extension events without the need for theaddition of polyethylene glycol.

FIG. 24 shows RPA using uvsX(C). In this experiment, uvsX(C) has beencombined with gp32(N) in the presence of the oligonucleotides Tester3bioand Sizer2. The template DNA in this experiment was the EcoRV digestedplasmid carrying the E. coli ruvB gene used in Example 1. The reactionsolution included 10 mM Mg-acetate, 27 mM phosphocreatine, 100 ng/μlcreatine kinase, 400 nM Tester3bio, 400 nM Sizer2, 3 mM ATP, 200 μMdNTPs, 50 U Klenow fragment, 10 U chicken myokinase, 10 μg (1×) or 20 μg(2×)C-tag uvsX, 8.8 μg gp32(N). Optionally, we included 0.2 mM ADP (3S,10 μg E. coli topoisomerase 1,10% PEG 1450, and 10 μg uvsXA21(C).Tester3bio recognised one end of an approximately 300 base pair fragmentand included a 5′ overhang relative to the end of the target. Sizer2recognised the other strand of this template and was directed toward anembedded sequence such that its 3′ end is about three and a half helicalturns from the end of Tester3bio.

In the presence of PEG 1450, we observed the amplification of theexpected fragment within 2 hours. In the cases where amplificationoccurred, almost the entire of population of oligonucleotides wasconsumed indicating an amplification of 3-5×10⁴. The reaction componentsare indicated. Included in some samples are additional components. Wefound that 200 μM ADP-β-S slightly increased the amount of productgenerated under these conditions. Conversely under the conditions usedhere, addition of E. coli toposiomerase I inhibited amplification. Underthe conditions used, we detected no amplification with uvsXA21(C)protein. However, no PEG1450 was included in these samples, and uvsX(C)did not amplify either under these conditions without PEG 1450.

FIG. 25 shows wild-type versus modified gp32. The variant uvsX(C)protein was determined to be qualitatively different to native untaggedgp32. The reaction solution included 10 mM Mg-acetate, 27 mMphosphocreatine, 100 ng/μl creatine kinase, 300 nM Tester3bio, 300 nMSizer2, 3 mM ATP, 200 μM dNTPs, 50 U Klenow fragment, 10 U chickenmyokinase, 300 ng/μl uvsX(C), 300 ng/ml gp32 (untagged) or 100, 200,300, 400, 500, or 600 ng/ml gp32(C), and PEG as indicated. The reactionwas incubated for 2 hours at 37° C. A comparison is shown betweenamplification reactions performed in the presence of untagged gp32 and atitration of gp32(C), either in the presence or absence of PEG1450. Weobserved that PEG was required in the reaction for gp32(C) to function,while this is not the case for untagged gp32. However, PEG significantlyincreased the quantity of product formed during the reaction period ineither case. Even in the presence of PEG, untagged gp32 consistentlyappeared to generate slightly more product than gp32(C) at each point onthe titration curve.

FIG. 26 shows titration of gp32 and effect of uvsY. Titration of gp32revealed a requirement for a minimal quantity of gp32, and a requirementfor uvsY(N) protein when untagged gp32 was employed. In FIG. 26A, theresults of an experiment are shown demonstrating that when untagged gp32was used, uvsY(N) protein was required for amplification. The reactionsolution included 10 mM Mg-acetate, 27 mM phosphocreatine, 100 ng/μlcreatine kinase, 300 nM Tester3bio, 300 nM Sizer2, 3 mM ATP, 200 μMdNTPs, 10% PEG1450, 50 U Klenow fragment, 10 U chicken myokinase, 300ng/μl uvsX(C), 300 ng/μl gp32 (untagged), and uvsY(N) as indicated inthe figure. The solution was incubated for 2 hours at 37° C. The uvs(Y)protein operated over a range of concentrations shown here (50 to 300ng/μl). Other experiments demonstrated that higher quantities inhibitedthe reaction. Thus, an optimum must be established for any givenreaction (probably between 5 and 50 ng/μl).

FIG. 26B shows the results of titrating the untagged gp32 protein in thepresence or absence of uvsY(N). The reaction solution included 10 mMMg-acetate, 27 mM phosphocreatine, 100 ng/μl creatine kinase, 300 nMTester3bio, 300 nM Sizer2, 3 mM ATP, 200 μM dNTPs, 10% PEG1450, 10 Uchicken myokinase, 750 ng/μl uvsX(C), 300 ng/μl gp32 (untagged), and 300ng/μl uvsY(N). The solution was incubated for 2 hours at 37° C. Onceagain, there is a requirement for uvsY(N) to achieve amplification.Additionally, this experiment demonstrates a need for a minimal amountof gp32. In this experiment, varying the gp32 concentrations between 80and 160 ng/μl gp32 resulted in a sharp transition from no amplificationto efficient amplification. A simple analysis of known binding site sizefor gp32, the length of the oligonucleotides, and their concentration,suggested that this rise in concentration would represent a transitionfrom substoichiometric levels of gp32 for the primer, to saturatinglevels. Consequently, the simplest interpretation is that gp32 saturatesthe oligonucleotides in order to have excess gp32 in the reaction tocollect and stabilise the outgoing strand.

FIG. 27 shows factors affecting reaction rate and noise. High gp32cooperativity inhibits recombinase filament formation (FIG. 27A). Inaddition, uvsY acts to increase recombinase loading in an unfavorablegp32 environment (FIG. 27B). PEG aids the function ofcooperatively-disabled gp32 (FIG. 27C). The reaction rate is influencedby both recombinase activity and effectiveness of gp32 (FIG. 27D).Post-invasion phases are enhanced in the presence of cooperative gp32(FIG. 27E). Cooperative gp32 is more effective at silencing reactionnoise (FIG. 27F).

The predicted effects and interactions of gp32, uvsX, uvsY, and PEG weresuggested, with the conclusion that an optimal balance between reactionrate and noise must be reached. The degree of cooperativity of gp32 isindicated across the top of the figure. High cooperativity favouredefficient binding to single-stranded DNA, which prevented significantreaction noise by inhibiting undesirable priming behaviour. Highcooperativity also favoured stabilisation of the outgoing strand duringrecombination and DNA synthesis. Conversely highly cooperative gp32reduced the abundance of recombinase-loaded searching filaments, and canaffect reaction rate considerably.

This behaviour could be partly overcome by including uvsY in thereaction. However, whether this could achieve as high a loading rate asdesired is yet to be determined. One could employ modified gp32 proteinsthat are less cooperative. Also, the cooperativity of gp32 proteins anduvsX proteins can be affected by the inclusion of PEGs. PEG may alsohave beneficial, or sometimes detrimental, consequences on othercomponents of the reaction such as DNA hybridisation behaviour andpolymerase processivity. Optimal rate may be acquired at some positionaway from either extreme, as indicated, as a balance between recombinaseloading and gp32 function may need to be reached.

FIG. 28 shows the effects of PEG. PEG was able to reduce the averagelength of linear invasion/run-on products in uvsX-mediated linear run-onexperiment in the presence of gp32(C). Shown are the results of a linearrun-on experiment utilising the Tester3bio oligonucleotide targeted tothe end of the approximately 300 base pair EcoRV fragment of E. coliRuvB. This fragment was used throughout the disclosed experiments and isdepicted schematically on the right of the figure. Reaction werereconstituted with 8 mg of UvsX(C) in a final reaction volume of 30 ml,in the presence of gp32(C), and in some cases, varying amounts ofUvsY(N) or UvsY(C). The reaction solution included 10 mM Mg-acetate, 27mM phosphocreatine, 1 U creatine kinase, 0.4 μM Tester3bio, 3 mM ATP,200 μM dNTPs, 50 U Klenow fragment, 1 U chicken myokinase, 8 μg uvsX(C),7.5 μg gp32(C) or 8.8 μg gp32(N), and 0.5 pmoles template. The finalvolume was 30 μl. The solution was incubated for 2 hours at 37° C., and2 μl of the solution was loaded in each lane.

In the absence of PEG, multiple invasion/run-on cycles appear to haveoccurred on each template, and there was generation of a significantamount full-length product. However, an even greater amount of slightlyshorter fragment was produced, which we believe constitute slightlyshorter run-ons which may have folded back on themselves and synthesiseda short hairpin. We interpreted all bands not full-length as resultingfrom some form of bona fide invasion/extension reaction that has notachieved full extension. Both UvsY(N) and UvsY(C) stimulate the quantityof product formed to some extent. In the case of no PEG, UvsY(C) seemsto be more effective than UvsY(C). This is in contrast to othergeometric amplification data we generated, suggesting that only UvsY(N)supported efficient geometric amplification.

Most notably, the inclusion of PEG, in contradiction to findings with E.coli recA, seemed to decrease the overall amount of product on this gel.It also decreased the average length distribution of products. In orderto explain this, we suggest that under these conditions thecooperativity of gp32(C), perhaps already at a maximum, cannot beincreased by PEG whilst that of UvsX can be. Thus, relativelyhyperactive UvsX behaviour results in rapid loading onto the outgoingstand, reinvasion, and efficient ‘bubble migration’ which chases thenewly synthesised strand and displaces it more readily. Consequently,the average product length is significantly reduced.

FIG. 29 shows DNA end directed invasion. The first round ofinvasion/synthesis using end-targeting and oligonucleotide overhang isillustrated (FIG. 29A). Additional ˜10 to 15 residues (5′ end) and ˜30residues (3′ end) approximate the minimal requirement for strandexchange (FIG. 29B). Invasion occurs, followed by release of thedeconstrained outgoing strand, and backfire synthesis (FIG. 29C). Mostnucleoprotein filaments that are coated enough to complete strandexchange will catalyze complete and deconstrained release of theoutgoing strand (FIG. 29D). Subsequent rounds of invasion/synthesisoccur (FIG. 29E). Few or no nucleoprotein filaments can exchange to theend of the target (FIG. 29F). One or no gp32 molecules are provided(FIG. 29G). Following this is recombinase loading (FIG. 29H) and branchmigration (FIG. 29I).

This figure describes how targeting oligonucleotides initiallypossessing an overhang relative to a linear target template might behaveduring the first and then subsequent attempts to carry out strandexchange. The purpose of this model is to rationalise data suggestingthat when such a situation in reconstituted experimentally, there is asignificant difference between the first and subsequent invasions. Thetop of the figure depicts recombinase loaded oligonucleotide filaments,displaying different 5′ extents of coverage. The 5 to 3′ directionalassembly means that most should have coating to the very 3′ end. Asdepicted in the figure, the oligonucleotides all possess a 5′ overhangrelative to the initial target.

The first invasion event is likely to result in complete release of theoutgoing strand as there is a significant likelihood that recombinasewill coat the searching oligonucleotide to a further 5′ extent than thesequence that will be involved in the strand exchange. Once the outgoingstrand is freed, it is topologically unconstrained and can be easilystabilised by single-stranded DNA binding proteins, presumably eventhose with relatively poor cooperativity. Furthermore, stability is alsogenerated by polymerases extending the 3′ end of the duplex DNA togenerate the complement to the very 5′ end of the targetingoligonucleotide. We refer to this synthesis as backfire synthesis. As aconsequence of backfire synthesis any subsequent invasion will be flushwith the extended template.

Under these circumstances, most oligonucleotides are not completelycoated with recombinase to the their very 5′ ends. In some cases, theremay be one or more gp32 molecules coating the 5′ part of theoligonucleotide. When these oligonucleotides perform strand exchange onthe now extended target, the outgoing strand is unlikely to beimmediately freed. As a consequence, the event initially resembles thetopologically constrained event already depicted in FIG. 6. The modelsuggests that only if, the cooperativity of the single-stranded DNAbinding protein is sufficient will these strained unstable intermediatesbe able to exist for some limited period. In the bottom part of thefigure, we explore what might occur to these unstable intermediates.

In scenario 1, the unexchanged 5′ extent of the oligonucleotideundergoes branch migration with the equivalent duplex portion of thetarget. This could easily lead to complete dissociation of this part ofthe outgoing strand which would then rapidly rotate to release anystress and be stabilised by single-stranded DNA binding proteins asoccurred in the first invasion. These now stable substrates will beideal and relatively stable assemblies for polymerase elongation.Alternatively, in scenario 2, the single-stranded DNA binding proteindisassembles from the outgoing strand and branch migration proceeds inthe opposite direction to that in scenario 1, so that the invading DNAis ejected. In scenario 3, the outgoing strand becomes coated withrecombinase and re-invades leading to ejection of the oligonucleotide.This process resembles a process described elsewhere as bubblemigration. If recombinase loads onto the freed outgoing strand inscenario 1 then bubble migration could also occur. We have experimentaldata that is most easily reconciled by considering the existence ofbubble migration as shown in FIG. 28.

FIG. 30 shows RPA in a complex sample. In this experiment we haveexamined the sensitivity of RPA, and need for a DNA-end, in an RPAreaction on a complex DNA target. A schematic representation is given ofthe DNA sequence, which corresponds to part of the human angiotensinconverting enzyme (ACE) gene. Three different combinations of primerswere used.

The experiment used a mixture of uvsX(C), uvsY(N), and gp32(C)K3A. Thereaction solution included 10 mM Mg-acetate, 27 mM phosphocreatine, 100ng/μl creatine kinase; 300 nM Up3 primer; 300 nM Down1, 2, or 3 primer;3 mM ATP, 200 μM dNTPs, 10% PEG1450, 50 U Klenow fragment, 10 U chickenmyokinase, 300 ng/μl uvsX(C), 300 ng/μl gp32(C)K3A, and 50 ng/μluvsY(N). The final volume was 30 μl. The solution was incubated for 5hours at 37° C. Reaction products were electrophoretically separated onan acrylamide gel, transferred to a nylon membrane, and probed with abiotinylated oligonucleotide recognising a unique internal sequence.

For the RPA reaction, uncut template (genomic DNA) and cut template werecompared, and primer pairs were compared. A fragment of the expectedsize was detected. In all cases, there was no specific product when nogenomic DNA is added to the reaction, but a specific product wasgenerated when DNA (equivalent to roughly 10,000 copies of any sequence)was added. Digestion of the DNA prior to RPA with HpaII resulted in thegeneration of at least one end overlapping with one of theoligonucleotides, and an increase in signal strength. However, there wasnot an absolute requirement for HpaII digestion for RPA to occur.

FIG. 31 shows RPA sensitivity. In this experiment, we have examined thesensitivity in an RPA reaction on a complex DNA target. A schematicrepresentation is given of the DNA sequence, which corresponds to partof the human angiotensin converting enzyme (ACE) gene. Three differentcombinations of primers were used. The experiment used a mixture ofuvsX(C), uvsY(N), and gp32(C)K3A. The reaction solution included 10 mMMg-acetate, 27 mM phosphocreatine, 100 ng/μl creatine kinase; 300 nM Up3primer; 300 nM Down1, 2, or 3 primer; 3 mM ATP, 200 μM dNTPs, 10%PEG1450, 50 U Klenow fragment, 10 U chicken myokinase, 300 ng/μluvsX(C), 300 ng/μl gp32(C)K3A, and 50 ng/μl uvsY(N). The final volumewas 30 μl. The solution was incubated for 5 hours at 37° C., and probeACE-hyb was used. Reaction products were electrophoretically separatedon an acrylamide gel, transferred to a nylon membrane, and probed with abiotinylated oligonucleotide recognising a unique internal sequence. Afragment of the expected size was detected. In all cases, there was nospecific product when no genomic DNA is added to the reaction, but aspecific product was generated when sufficient DNA was added. In allcases, 1000 copies were sufficient to generate a significant signal, andin one case we could detect a very faint signal at 100 copies.

FIG. 32 shows RPA sensitivity and template independent artifacts. Theresults of an experiment are shown in which we have investigated thesensitivity in an RPA reaction on a complex DNA target. A schematicrepresentation is given of the DNA sequence, which corresponds to partof the human angiotensin converting enzyme (ACE) gene. A time course ofthe amplification was performed taking reaction samples at 1, 2 and 3hours. Reaction products were detected by virtue of the presence of abiotin residues attached to the 5′ end of one of the oligonucleotidesused in the amplification. In this way, it was possible to visualize allthe reaction products involving this oligonucleotide, including anyartifacts that might arise. We tested several different concentrationsof the uvsY(N) protein. The reaction solution included 10 mM Mg-acetate,27 mM phosphocreatine, 100 ng/μl creatine kinase; 300 nM Up3 primer; 300nM Down1 primer; 3 mM ATP, 200 μM dNTPs, 10% PEG1450, 50 U Klenowfragment, 10 U chicken myokinase, 300 ng/μl uvsX(C), 300 ng/μl gp32(C),and 50 ng/μl uvsY(N). The final volume was 30 μl. The solution wasincubated for 5 hours at 37° C. At an uvsY(N) concentration of 50 ng/μl,we detected the correct product directly, albeit faintly, after 3 hoursof incubation. During this period there was an accumulation of strongbands of roughly twice the length of the oligonucleotide, whichaccumulated similarly in the template minus sample. These were mostlikely to be primer artifacts.

FIG. 33 shows how primer artifacts may arise. Primer artifacts likelyinitiate by erroneous self-priming events as depicted here. Primers mayform hairpins, as occurs in FIG. 33A, or hybridise to a second primer,as occurs in FIG. 33B. If a polymerase can extend such a hairpin asignificant stretch of double-stranded DNA may be formed, as seen in A*and B*. These structures might become targets for otherrecombinase-loaded filaments, titrate active filaments from bona fidetargets, and possibly enter into geometric forms of amplificationthemselves.

FIG. 34 shows primer artifact suppression. Depicted schematically areseveral strategies to suppress primer artifact noise. In FIG. 34A, asecond short, 3′-blocked oligonucleotide complementary to the 3′sequences of the targeting oligonucleotide is included in the reactionto compete for the formation of secondary structure formation that mightresult in erroneous priming. In FIGS. 34B and 34C, a similar shortblocked oligonucleotide is employed as in (A), but in this case acovalent bridge is engineered between the 5′ end of the targetingoligonucleotide and the 5′, or 3′, end of the competing short primer. Inthis way, the blocked nucleotide is tethered at its 5′ end to the 5′ endof the targeting oligonucleotide (FIG. 34A-B) In FIG. 34D, a shortsequence complementary to the 3′ region of the targeting oligonucleotideis added to the 5′ region of the targeting oligonucleotide. It competesefficiently with secondary structure formation.

FIG. 35 shows use of hairpin oligonucleotides to stimulate self-primingof displaced strands. Depicted is a scheme showing how oligonucleotideswhose design includes a 5′ section with perfect complementarity to the3′ section might be used to stimulate amplification throughself-priming. At the top of the diagram is shown a target DNA,designated A, which has distinct ends which are targets for one of thetwo targeting oligonucleotides shown in the upper left and right of thefigure. Both of these oligonucleotides possess complementarity betweentheir 5′ and 3′ regions as indicated by short arrows. The target, A,would likely have been generated by earlier invasion/elongation eventsinvolving these oligonucleotides and an initial target lacking the5′-most regions of these oligonucleotides. On the left or right side ofthe figure we follow the outcome of invasion/elongation events initiatedby targeting by the left or right primers respectively. The outcome issimilar in both cases, albeit the final products are arranged slightlydifferently.

Focusing on the left side of the figure we observe that when target A issubject to invasion and elongation with the left primer the result isformation of a new duplex identical to A and a single-stranded DNAequivalent to the top strand of the initial target, designated B. Due tothe presence of the complementarity between the very 3′ region andadjacent sequences, B is capable of forming a hairpin which will primeDNA synthesis to generate a largely double-stranded product C. Theproduct C can readily be targeted once again by the leftoligonucleotide. However, in this case, no single-stranded displacedstrand is formed. Instead, product D is formed with a length that isroughly twice that of the original target. This product is an invertedrepeat and contains two sequences that are targets for the leftoligonucleotide, and one for the right oligonucleotide located in themiddle.

Subsequent invasion/elongation events, and the possible occurrence ofhybridisation events between displaced strands, could easily lead tosuch ‘dimeric’ species becoming further enlarged, and the formationgenerally of more complex products. A similar course of events is shownon the right side of the figure, this time initiated byinvasion/elongation by the right primer. The final dimeric product, D,is not equivalent to D as the two end sequences are targets for theright primer, and the central region is a target for the left primer.Processes similar or identical to those shown here are likely to occurwith some frequency under some conditions even in the absence of thedeliberate design of oligonucleotides to promote it, as there is oftensome limited capacity for self-priming of single-stranded DNAs.

FIG. 36 shows conditions that support the amplification of DNA withlittle or no primer artifacts. The results of an experiment are shown inwhich we have investigated the sensitivity of an RPA reaction on acomplex DNA target. A schematic representation is given of the DNAsequence, which corresponds to part of the human angiotensin convertingenzyme (ACE) gene. Oligonucleotides used are the biotinylated Angio1bioprimer and the unbiotinylated Angio3 primer whose sequence is given inthe experimental methods. These primers amplified a 132 bpdouble-stranded DNA fragment. Uncut human genomic DNA was titrated from45 copies up to 2880 copies. Reaction products were detected by virtueof the presence of a biotin residue attached to the 5′ end of one of theoligonucleotides used in the amplification. In this way, it was possibleto visualize all the reaction products involving this oligonucleotide,including any artifacts that might arise.

The reaction was incubated for 2 hours at 37° C. in the case of theKlenow exo-, and 2 hours at 42′C in the case of the Bst polymerase. Thereaction included the following: 50 ng/μl uvsY(N), 300 ng/μl gp32(C),100 ng/μl uvsX(C), 20 mM phosphocreatine, 3 mM ATP, 25 milliunits/μlmyokinase, 100 ng/μl creatine kinase, 200 μM dNTPs, 5% w/v PEG compound,300 nM Angio1bio primer, 300 nM Angio3 primer, 800 ng/μl Klenow exo- or1.2 units/μl Bst polymerase. The Klenow-mediated amplification wasperformed in U2 buffer comprising a final composition of 20 mM Trisacetate pH 7.9, 8 mM magnesium acetate, 120 mM potassium acetate. TheBst polymerase-mediated amplification was performed in U1 buffercomprising a final composition of 20 mM Tris acetate pH 7.5, 6 mMmagnesium acetate, 100 mM potassium acetate.

Example 13 DNA Amplification for Point-of-Use Applications

Clones and proteins were produced as described in Example 11, above.

DNAs Used in RPA Reactions

We have employed several different target DNAs in this study, and anumber of oligonucleotides. The sequence of the oligonucleotides isgiven below, and the template target in the experiment shown in FIG.39B. The E. coli RuvB gene target was used for linear run-on assay (FIG.39B). Identical quantities of plasmid template containing this fragmentwere cut either with EcoRV, releasing a roughly 300 bp fragment, or withClaI which linearises the DNA. Equal molar quantities of template wereused in the run-on experiments (20 nM each template). The sequence of aKpnI/ClaI fragment of this template is given below. The EcoRV fragmentis embedded within this sequence, and the sites are highlighted.

(SEQ ID NO: 18) GGTACCACTTTGCCGGAAGATGTAGCAGATCGCGCCATTCGCCCCAAATTACTGGAAGAGTATGTTGGTCAGCCGAGGTTCGTTCACAGATGGAGATTTTCATCAAAGCAGCGAAACTGCGCGGCGATGCCCTCGATCATTTGTTGATTTTTGGTCCTCCGGGGTTGGGTAAAACTACGCTTGCCAACATTGTCGCCAATGAAATGGGCGTTAATTTACGCACGACTTCTGGTCCGGTGCTGGAAAAGGCGGGCGATTTGGCTGCGATGCTCACTAACCTTGAACCGCATGACGTGCTGTTTATTGATGAGATCCACCGTCTATCGCCAGTTGTTGAAGAAGTGCTGTACCCGGCAATGGAAGACTACCAACTGGATATCATGATTGGTGAAGGTCCGGCGGCACGCTCCATTAAAATTGATTTGCCGCCGTTTACCCTGATTGGTGCAACCACGCGCGCAGGTTCGCTGACATCACCGTTGCGCGACCGTTTTGGTATTGTGCAACGTCTGGAGTTTTATCAGGTGCCGGATCTGCAATATATCGTCAGTCGCAGCGCACGCTTTATGGGGCTTGAGATGAGTGATGACGGCGCGCTGGAAGTTGCTCGTCGCGCTCGCGGTACGCCGCGCATTGCCAACCGTCTGCTGCGTCGAGTGCGTGATTTCGCCGAAGTGAAGCACGATGGCACCATCTCGGCAGATATCGCTGCTCAGGCGCTGGATATGTTGAATGTCGATGCTGAAGGTTTCGATTATATGGACCGCAAATTGTTGCTGGCGGTAATCGAT

Oligonucleotide Tester3bio sequence. Bases homologous to the target arein bold.

(SEQ ID NO: 19) 5′biotin-CTCACTATACCTCAGCATCATGATTGGTGAAGGTCCG GCGGCAC

Human DNAs

We have used human genomic DNAs from several sources. A mixed populationmale genomic DNA from Promega was utilised. Also, DNA from individualmale samples was studied in FIG. 38A. Individual 1 and 2 were father andson. Individual 2 DNA was used in the experiment in FIG. 39E, while DNAfor the experiment in FIG. 39F was another male individual. Sequences ofoligonucleotides used for amplifying human and B. subtilis sequences areas follows:

Corresponding to the human ApolipoproteinB locus:

ApoB4 (SEQ ID NO: 20) 5′ CAGTGTATCTGGAAAGCCTACAGGACACCAAAA 3′ Apo300(SEQ ID NO: 21) 5′ TGCTTTCATACGTTTAGCCCAATCTTGGATAG 3′ Apo700(SEQ ID NO: 22) 5′ TGGTAAACGGAAGTCTGGCAGGGTGATTCTCG 3′ Apo800(SEQ ID NO: 23) 5′ CAATTGTGTGTGAGATGTGGGGAAGCTGGAAT 3′ Apo900(SEQ ID NO: 24) 5′ GAGGTGGTTCCATTCCCTATGTCAGCATTTGC 3′ Apo1000(SEQ ID NO: 25) 5′ GGGTTTGAGAGTTGTGCATTTGCTTGAAAATC 3′ Apo1500 (SEQ ID NO: 26) 5′ TTGAATTTCAAGTTTAGAAAAGTTGAGGGAGCCAG 3′

Corresponding to the human SRY locus:

SRY3  (SEQ ID NO: 27) 5′ AAAGCTGTAACTCTAAGTATCAGTGTGAAAC 3′ SRY4 (SEQ ID NO: 28) 5′ GTTGTCCAGTTGCACTTCGCTGCAGAGTACC 3′

Corresponding to B. subtilis genomic DNA:

BSA1 (SEQ ID NO: 29) 5′ TTGGGCACTTGGATATGATGGAACTGGCAC 3′ BSA3(SEQ ID NO: 30) 5′ ACAGAAAGCTATTAAAGCAACTGACGGTGTGG 3′ BSB3(SEQ ID NO: 31) 5′ CCATCTTCAGAGAACGCTTTAACAGCAATCC 3′

Human STR marker primers:

CSF1PO  (SEQ ID NO: 32) 5′ GTTGCTAACCACCCTGTGTCTCAGTTTTCCTAC  CSF1PO (SEQ ID NO: 33) 3′ AGACTCTTCCACACACCACTGGCCATCTTCAGC  D7S820(SEQ ID NO: 34) 5′ GAACACTTGTCATAGTTTAGAACGAACTAACG  D7S820 (SEQ ID NO: 35) 3′ GAATTATAACGATTCCACATTTATCCTCATTGAC  D13S317 (SEQ ID NO: 36) 5′ TTGCTGGACATGGTATCACAGAAGTCTGGGATG  D13S317 (SEQ ID NO: 37) 3′ CCATAGGCAGCCCAAAAAGACAGACAGAAAGA  D16S539 (SEQ ID NO: 38) 5′ AAACAAAGGCAGATCCCAAGCTCTTCCTCTTCC  D16S539 (SEQ ID NO: 39) 5′ ATACCATTTACGTTTGTGTGTGCATCTGTAAGC  D18S51 (SEQ ID NO: 40) 5′ GGTGGACATGTTGGCTTCTCTCTGTTCTTAAC  D18S51 (SEQ ID NO: 41) 3′ GGTGGCACGTGCCTGTAGTCTCAGCTACTTGC  THO1 (SEQ ID NO: 42) 5′ TACACAGGGCTTCCGGTGCAGGTCACAGGGA  THO1 (SEQ ID NO: 43) 3′ CCTTCCCAGGCTCTAGCAGCAGCTCATGGTGG  TPOX (SEQ ID NO: 44) 5′ ACTGGCACAGAACAGGCACTTAGGGAACCC  TPOX  (SEQ ID NO: 45)3′ GGAGGAACTGGGAACCACACAGGTTAATTA 

Timecourse experiment:

APOB600  (SEQ ID NO: 46) GCTCACTGTTCTGCATCTGGTCAATGGTTCTG APOB300REV (SEQ ID NO: 47) CTATCCAAGATTGGGCTAAACGTATGAAAGCA

Shorter oligonucleotide experiment:

APOB500  (SEQ ID NO: 48) ATGGTAAATTCTGGTGTGGAAAACCTGGATGG  APO500-28 (SEQ ID NO: 49) TAAATTCTGGTGTGGAAAACCTGGATGG  APO500-25  (SEQ ID NO: 50)ATTCTGGTGTGGAAAACCTGGATGG  APOB300REV  (SEQ ID NO: 51)CTATCCAAGATTGGGCTAAACGTATGAAAGCA  APOB300REV-28  (SEQ ID NO: 52)CCAAGATTGGGCTAAACGTATGAAAGCA  APOB300REV-25  (SEQ ID NO: 53)AGATTGGGCTAAACGTATGAAAGCA  D18S51 5′ (SEQ ID NO: 54)GGTGGACATGTTGGCTTCTCTCTGTTCTTAAC  D18S51 5′-28  (SEQ ID NO: 55)GACATGTTGGCTTCTCTCTGTTCTTAAC  D18S51 5′-25  (SEQ ID NO: 56)ATGTTGGCTTCTCTCTGTTCTTAAC  D18S51 3′ (SEQ ID NO: 57)GGTGGCACGTGCCTGTAGTCTCAGCTACTTGC  D18S51 3′-28  (SEQ ID NO: 58)GCACGTGCCTGTAGTCTCAGCTACTTGC  D18S51 3′-25  (SEQ ID NO: 59)CGTGCCTGTAGTCTCAGCTACTTGC  SRY3  (SEQ ID NO: 60)AAAGCTGTAACTCTAAGTATCAGTGTGAAAC  SRY3-28  (SEQ ID NO: 61)GCTGTAACTCTAAGTATCAGTGTGAAAC  SRY3-25  (SEQ ID NO: 62)GTAACTCTAAGTATCAGTGTGAAAC  SRY4  (SEQ ID NO: 63)GTTGTCCAGTTGCACTTCGCTGCAGAGTACC  SRY4-28  (SEQ ID NO: 64)GTCCAGTTGCACTTCGCTGCAGAGTACC  SRY4-25  (SEQ ID NO: 65)CAGTTGCACTTCGCTGCAGAGTACC 

Conditions of standard RPA reactions included: 50 mM Tris pH 8.4, 80 mMPotassium acetate, 10 mM Magnesium acetate, 1 mM DTT, 5% PEG compound(Carbowax-20 M), 3 mM ATP, 20 mM Phosphocreatine, 100 ng/μl Creatinekinase, 600 ng/μl gp32; 109 ng/μl, or 125 ng/μl, or 200 ng/μl uvsX; 16ng/μl, or 25 ng/μl, or 40 ng/μl, or 60 ng/μl uvsY; 20 ng/μl Bsupolymerase, 200 μM dNTPs, and 300 nM each oligonucleotide. Reactionconditions C1-C4 are as above with: C1=109 ng/μl uvsX, 16 ng/μl usvY;C2=125 ng/μl uvsX, 25 ng/μl uvsY; C3=200 ng/μl uvsX, 40 ng/μl uvsY;C4=200 ng/ml uvsX, 60 ng/μl uvsY.

Experimental Results

FIG. 37 shows a schematic representation of RPA method. In FIG. 37A(i),Recombinase protein uvsX binds cooperatively to single-strandedoligonucleotides in the presence of ATP. Nucleoprotein filamentsactively hydrolyse ATP to ADP. Spontaneous disassembly can lead tocompetitive binding of single stranded binding protein gp32, this beingdeterred and reloading aided by uvsY protein and polyethylene glycol. InFIG. 37A(ii), recombinase filaments catalyse strand exchange ifhomologous DNA is detected. In FIG. 37A(iii), strand exchange matchesthe searching strand with its complement and displaces a strand thenbound by gp32. Recombinase disassembles. In FIG. 37A(iv), polymerasesaccess the structure and extend the oligonucleotide, displacing more ofthe original strand. In FIG. 37B(i), two opposing targetingnucleoprotein complexes recombine with their respective targets and DNAsynthesis is initiated. In FIG. 37B(ii), polymerase complexes encounterone another and one of the polymerases dissociates. In FIG. 37B(iii),the remaining polymerase continues synthesis freeing the two parentstrands, a polymerase re-binds to the free 3′-end thus replication ofboth strands occurs. In FIG. 37B(iv), new targeting events occur. In thesecond round one targeting primer will displace a free end. In FIG. 37C,comparison of the products of strand exchange at a DNA end or at anembedded DNA sequence.

FIG. 38A shows the results of amplification of STR markers from twoindividuals (1 and 2, father and son) using primer pairs for sevenindependent markers. RPA conditions C4 were employed (see above). FIG.38B=shows titration of reaction components to determine concentrationsthat support in vitro amplification. Reactions included the primers SRY3and SRY4 at 0.3 μM (targeting the SRY gene), 80 mM potassium acetate, 50mM TrisCl pH 8.4, 2 mM DTT, 5% Carbowax-20M, 200 ng/μl uvsX, 60 ng/μluvsY, 600 ng/μl gp32, 20 ng/μl Bsu polymerase, and 50 copies/μl Ychromosomal DNA, except when a given component was that underinvestigation. Optimal quantities of gp32, ATP, uvsX, uvsY, PEG, and Bsupolymerase for effective amplification of this particular product weredetermined. ATP-γ-S and ADP-β-S inhibited the reactions.

FIG. 39A shows the size limits of RPA reactions. Primer ApoB4 wascombined with opposing primers capable of generating amplified productsof the indicated sizes. Conditions of 125 ng/μl uvsX and 25 ng/μl uvsY(C2) were employed except C1 where 109 ng/μl uvsX and 16 ng/μl uvsY wereused; 15 copies/μl human DNA were used (30 μl reactions). Underconditions C2, some hairpin-mediated product duplication occurredconverting some of the 300 bp amplicon to 2× and 3× unit length (*) (L.D. Harris, J. D. Griffith, J Mol Biol 206, 19-27 (Mar. 5, 1989)).

FIG. 39B shows elongation efficiencies from embedded or end sequences. Abiotinylated primer was incubated with linearized plasmid DNA. Equalquantities (20 nM final) of templates linearized with either ClaI (lane3) or EcoRV (lanes 1 and 2) were used, the primer either overlapping thecut end, or the target site being embedded (lane 3). Incubation withrecombinase targeting components with (lanes 2 and 3) or without(lane 1) Klenow reveals limited elongation from the embedded site(product 1*), and abundant elongation from the end site (product 2*).Electrophoresed products were transferred to nylon and biotin detectedby chemiluminescence. The weak common band (˜300 bp, lanes 1 and 3) wasan artifact arising from this particular protocol.

FIG. 39C shows the sensitivity of RPA reactions. The indicated copynumber of B. subtilis genomes was amplified with oligonucleotides BsA1and BsB3, which amplify a 200 bp fragment. Conditions C1 were employed.FIG. 39D shows human DNA of the indicated copy number amplified withprimers ApoB4 and Apo300 to generate a 300 bp fragment. Conditions C2were employed. FIG. 39E, F show results from human DNA from singleindividuals. The DNA was diluted and samples theoretically containingthe indicated copy number were amplified with primers D 18S51 5′ and 3′which amplify an STR of size ˜300-360 bp. At predicted copy numbers of 2or 3, a number of samples amplified single alleles (*). Conditionsemployed were C2 in (E), and C4 in (F).

FIG. 40 shows specificity of RPA reactions. Primers BsA3 and BsB3, whichamplify a 380 bp fragment from B. subtilis genomic DNA were incubatedwith 1 μg of human DNA, with (+) or without (−) addition of 100 copiesof B. subtilis DNA (FIG. 40A). An asterisk indicates the position of theexpected reaction product, and an arrow indicated the position of thegenomic DNA. Conditions C3 were employed. To investigate how long ittakes RPA to generate detectable reaction products a series ofamplification reactions were established with oligonucleotides Apo600bioand Apo300rev generating a 345 bp fragment. A copy number of 60copies/μl (FIG. 40B) or 6 copies/μl (FIG. 40C) were used. Individualreactions were stopped at the indicated number of minutes and analysedon a gel. Conditions C4 were employed (FIG. 40D).

For long-term storage of reaction components we lyophilised RPAreactions. Mixtures of reaction components were assembled in the absenceof the indicated components, PEG and buffer. The material wasfreeze-dried, and then reconstituted with PEG and buffer plus theadditional omitted components. Primers used are indicated. Allcomponents apart from PEG and buffer could be lyophilised andsuccessfully reconstituted in a functional reaction. Target DNA washuman male genomic DNA at 150 copies/μl (FIG. 40E). Oligonucleotidestargeting three independent loci in human genomic DNA were incubatedwith overlapping primer pairs of 25, 28, or 32 bases as indicated. Onlythe primers of 32 bases in length successfully amplified targets. Otherexperiments show that primers of 30 residues are also effective atamplifying target DNAs.

FIG. 41 shows primer noise at low target copy number.

Two primers, BsA1 and BsB3 (BsA1-5′TTGGGCACTTGGATATGATGGAACTGGCAC3′ (SEQID NO:29), BsB3-5′CCATCTTCAGAGAACGCTTTAACAGCAATCC3′ (SEQ IDNO:[[30]]31), which flank a roughly 300 bp fragment of the Bacillussubtilis genome were incubated with B. subtilis genomic DNA seriallydiluted with water. Conditions used were 80 mM Potassium acetate, 50 mMTris.Cl pH 8.4, 2 mM DTT, 5% Carbowax 20M, 200 μM dNTPS, 3 mM ATP, 20 mMPhosphocreatine, 50 ng/μl creatine kinase, 300 nM each oligonucleotide,800 ng/˜1 gp32, 120 ng/μl uvsX, 25 ng/μl uvsY, and 28 ng/μl Bsupolymerase. The reaction was incubated for 90 minutes at 37° C. Productswere separated on a 2.5% agarose gel and stained with ethidium bromide.Arrows indicate the expected position of the correct amplicon.

FIG. 42 details the selection of optimal primers by combining aselection of candidate forward and reverse primers and testing theoutcome at very low start copy densities.

Several primer pairs were designed to sequences in the Bacillus subtilissporulation locus Spo0B. The position and orientation of these primersis indicated in (B) as the following:

J1  SEQ ID NO: 66 ACGGCATTAACAAACGAACTGATTCATCTGCTTGG  J2  SEQ ID NO: 67ATAACCATTTCTTCAATCATTTCAAAGACACGGTC  K1  SEQ ID NO: 68TATAGACGCAAAGCACGAATCAAAGCTCTCAAACC  K2  residues 1-26 of SEQ ID NO: 69CCTTAATTTCTCCGAGAACTTCATAT  L1  SEQ ID NO: 70ATATGAAGTTCTCGGAGAAATTAAGGATTTGTCGG  L2  SEQ ID NO: 71AATCAGCTGTCTGTCAGGATGATCCGTTTGAAGCG  NEST1  SEQ ID NO: 72CATTAACAAACGAACTGATTCATCTGCTTGG  NEST2  SEQ ID NO: 73ACAGATGAAACAGCTTTCTCATCAGTTTCG.

These primers were combined as indicated in (A), and incubated for 90minutes under the following conditions: 80 mM Potassium acetate, 50 mMTris.Cl pH 8.4, 2 mM DTT, 5% Carbowax 20M, 200 μM dNTPS, 3 mM ATP, 20 mMPhosphocreatine, 50 ng/μl creatine kinase, 300 nM each oligonucleotide,800 ng/μl gp32, 120 ng/μl uvsX, 25 ng/μl uvsY, and 28 ng/μl Bsupolymerase, .about.2copies/μl B. subtilis genomic DNA (estimated byserial dilution), 37° C. Arrows indicated the position of productsdetermined to be the expected fragment based on size. Some primer pairsgive product, but it was accompanied by smearing, or some additionalbands. A few combinations fail to give product of the expected size. Theprimer J1 and K2 give a significant amount of the product of theexpected size, and rather cleanly. Interestingly when NEST1 primer,lacking the 4 5′-most bases present in J1, replaces J1 in the equivalentreaction, the reaction is not improved indicating that adding as well asremoving bases may improve primer performance even when all tested areover 30 residues in length.

FIG. 43 shows a theoretical consideration of how primer noise initiates.

(A) An oligonucleotide is shown, and above it is an arrow, the headindicating the 3′-end of the oligonucleotide. The sequence was generatedarbitrarily, although 4 of the 5 most 3′ bases form a palindrome, whichmight well be avoided in practice. (B) It is assumed that most initialevents consist of the 3′ end folding transiently onto more 5′ sequencesto transiently form a Watson-Crick base pair. (C) Occasionally apolymerase successfully elongates this structure to give a longdouble-stranded hairpin with a turn at one end (labeled H). The sequenceand 5′-3′ direction of the reverse complement of the original isindicated by a pale arrow. (D) In strategy 1 it is assumed that a secondrecombinase-coated oligonucleotide of identical sequence targets thehomologous double-stranded body of the hairpin, thus unraveling it.Mismatches occur at the 3′ region of the invading oligonucleotidebecause the complement to this section was not correctly generated instep C. If a 4 base pair palindrome had not fortuitously been generatedthe mismatches would be even more severe than shown here. (E) Apolymerase, very occasionally, might extend the mismatched 3′ region ofthis intermediate to generate an inverted repeat with mismatches in themiddle. This structure would also resemble the product if two identicalprimers had formed a brief 3′ overlap resulting in formation of a primerdimer. These structures cannot readily enter geometric phaseamplification. (F) In strategy 2 the long hairpin formed in (C) isbriefly unzipped at one end. This step might be enhanced by recombinasebinding to dsDNA because some reports indicate that recA may bind todsDNA and promote melting of short duplexes. (G) The transiently melted3′-end (reverse complement to the original 5′ end of theoligonucleotide) folds back in a similar manner to (B) and forms astructure capable of elongation. (H) Polymerase elongation creates alarge hairpin. In this case one end contains an identical duplexsequence to the parent oligonucleotide. (I) Another parentoligonucleotide invades the perfect duplex target. (J) Polymeraseelongation of (I) generates a large inverted repeat with the indicatedstructure. Both ends contain perfect primer target sites and so thisstructure can amplify exponentially. Interestingly, the inverted repeatstructure means that if an invasion and elongation occurs from one end,the displaced strand will rapidly tend to fold back onto itself to formthe structure shown in (H). This product will require anotherrecombination event to become structure (J), and thus it may be thathigher recombination activity particularly benefits noise amplification,and is strongly impeded by lower recombination activity.

FIG. 44 shows several oligonucleotide design strategies to improvesignal to noise in RPA reactions

Shown are three general strategies to improve signal to noise ratios inRPA reactions by facets of oligonucleotide design. (1) Progressivelyshortened oligonucleotides may be tested. The assumption is thatshortening of an oligonucleotide progressively from the 5′ end leads toa drop in activity of the respective nucleoprotein filament, and thatthis influences the doubling time of noisy artifacts more rapidly thanthat of target. Low activity nucleoprotein filaments may also becombined effectively with more active ones in some cases. (2) Lockednucleic acids, or other modified sugars, may be included in the primerswith consequent effect on amplification behaviour. The structure of alocked nucleic acid (LNA) sugar is indicated. (3) Oligonucleotidesbehaviour may be improved by the addition of 5′ residues, possiblyunrelated to the target, which suppress noise via mechanisms such asdeterring snapback priming, or by altering nucleoprotein recombinationactivity. Sequences may be homopolymer stretches, or otherwise. Idealsequences may be determined empirically.

FIG. 45 shows the consequences of reducing primer length

(A) Schematic description of experimental design. Pairs ofoligonucleotides were synthesized to three human targets, and sets ofprimers were made in which the 5′-most residues were progressivelydeleted (full length primers have been described above: D18S51 5′GGTGGACATGTTGGCTTCTCTCTGTTCTTAAC (SEQ ID NO:54), D18S51 3′GGTGGCACGTGCCTGTAGTCTCAGCTACTTGC (SEQ ID NO:57), SRY3 5′AAAGCTGTAACTCTAAGTATCAGTGTGAAAC 3′ (SEQ ID NO:27), SRY4 5′GTTGTCCAGTTGCACTTCGCTGCAGAGTACC 3′(SEQ ID NO:28), APOB500ATGGTAAATTCTGGTGTGGAAAACCTGGATGG (SEQ ID NO:48), APOB300REVCTATCCAAGATTGGGCTAAACGTATGAAAGCA (SEQ ID NO:51). Shorter primers weremade by deleting 5′-most bases to give the final indicated length).Primer pairs of identical length were combined in amplificationreactions in the fashion indicated. (B) Amplification reactions wereperformed on human genomic DNA, present in the start reaction at 50copies/μl. The three targets, an STR marker D18S51II, part of the humanSRY gene, and the human Apolipoprotein B locus, were amplified withpairs of oligonucleotides. The oligonucleotides were progressivelytruncated from the 5′ end in the manner as described in (A), and thelength of both primers in the primer pairs used is indicated above therespective lane. It is evident that there is a fairly precipitous dropin amplification behaviour as oligonucleotides are shortened from justover to just under 30 residues. Consequently comparing oligonucleotideswith length variation of just over to just under 30 residues isgenerally likely to reveal optimal length to get sufficient specificamplification, but very little or no background. For example two 29-merswere sufficient to amplify the D18S51 II locus, and do so cleanly.Amplification conditions were: 50 mM Tris.Cl pH 8.3, 90 mM Magnesiumacetate, 2 mM DTT, 80 mM Potassium acetate, 200 μM dNTPs, 600 ng/μlgp32, 200 ng/μl uvsX, 60 ng/μl uvsY, 300 nM primers, 20 ng/μl Bsupolymerase, 50 copies/μl genomic DNA, 90 minutes at 37° C.

FIG. 46 shows how a 3′ LNA residues alter amplification behavior in RPA

Amplification of a part of the human ACE locus is shown usingcombination of regular oligonucleotides and counterparts in which the3′-most residue has an LNA sugar. (A) Schematic representation of therelationship of oligonucleotide primers to one another. LNA1 and DNA1are identical except that LNA1 contains a LNA sugar at the most 3′position. Likewise LNA2 and DNA2 differ similarly. (sequence of LNA/DNA1is: GCTCCTTGGACTGGTAGATGTCACACTTGTG—SEQ ID 74, sequence of LNA/DNA2 isGCCTTGGCTCTGCTGTGCGCATGTGACTTAGC—SEQ ID 75). (B) RPA reactions wereassembled with the indicated oligonucleotide primer combinations.Product levels are significantly reduced when both oligonucleotidescontain a 3′ LNA under standard reaction conditions. Howeversubstitution of only one oligonucleotide does not suppress the reactiontoo greatly (lanes 2 and 3). Note that the background pattern in lane 3resembles that in lane 4, while in 2 it is largely absent. This suggeststhat the background derives principally from the activities of a singleprimer species. Amplification conditions were: 50 mM Tris.Cl pH 8.3, 90mM Magnesium acetate, 2 mM DTT, 80 mM Potassium acetate, 200 μM dNTPs,800 ng/μl gp32, 200 ng/μl uvsX, 60 ng/μl uvsY, 300 nM primers, 20 ng/μlBsu polymerase, 50 copies/μl genomic DNA, 120 minutes at 37° C. (C)Increasing the levels of polymerase result in restoration of robustactivity when two LNA-modified oligonucleotides are used. LNA1 and LNA2,or DNA1 and DNA2, were combined in the presence of the indicatedconcentrations of polymerase. Notably the DNA primers displayed highactivity at the lowest used concentration, and if anything the productslooked less good as polymerase concentration was increased. Shortproducts, 50-100 base pairs, are notably also seen. Conversely the LNAprimers were only optimally active at higher polymerase concentrations.Less small products were seen, and those seen other than the expectedsize are likely product related (see below). Amplification conditionswere: 50 mM Tris.Cl pH 8.3, 90 mM Magnesium acetate, 2 mM DTT, 80 mMPotassium acetate, 200 μM dNTPs, 800 ng/μl gp32, 200 ng/μl uvsX, 60ng/μl uvsY, 300 nM primers, 43 to 1400 ng/μl Bsu polymerase, 100copies/μl genomic DNA, 120 minutes at 37° C. (D) LNA-modifiedoligonucleotides give little, if any, background when no target ispresent, but efficiently amplify bona fide product to high levels whenreaction conditions are optimized. A polymerase concentration of 200ng/μl Bsu polymerase was employed. Reactions were carried out with DNAprimers pairs, or LNA primer pairs, in the presence or absence oftarget. A faint band of the expected size was found in the negativecontrols indicating a contamination problem. Nevertheless both primerpairs amplified efficiently in the presence of target, but in itsabsence the DNA pair gave significant noisy smear, while the LNA pairgave no other product than the slight contamination. This indicates thatthe LNA primer pair may be able to cleanly distinguish between thepresence and absence of target in the sample, while the DNA primersgenerate primer-derived products in the absence of target. Amplificationconditions were: 50 mM Tris.Cl pH 8.3, 90 mM Magnesium acetate, 2 mMDTT, 80 mM Potassium acetate, 200 μM dNTPs, 800 ng/μl gp32, 200 ng/μluvsX, 60 ng/μl uvsY, 300 nM primers, 200 ng/μl Bsu polymerase, 50copies/μl genomic DNA, 120 minutes at 37° C.

FIG. 47 shows that adding homopolymeric stretches to the 5′ ends ofprimers may alter nucleoprotein activity.

(A) The sequence of six oligonucleotides used in the experiment isshown. The J1 and K2 oligonucleotide target the B. subtilis SpoOB locus(SEQ IDs 66 and 69 respectively), and the other listed oligonucleotidesare derivatives of them, carrying a homopolymeric stretch of cytosines,or guanosines as indicated. These oligonucleotides can be incubated in apairwise fashion to assess which pair most effectively amplifies thetarget. (B) The results of incubating the six primers alone, or in allpossible combinations, is shown. Reactions were incubated at 37° C. for2 hours. Reaction conditions were 50 mM Tris.Cl pH 8.4, 2 mM DTT, 5%Carbowax 20M, 80 mM Potassium acetate, 200 μM dNTPS, 3 mM ATP, 20 mMPhosphocreatine, 50 ng/μl creatine kinase, 300 nM each relevantoligonucleotide, 800 ng/μl gp32, 120 ng/μl uvsX, 25 ng/μl uvsY, and 28ng/μl Bsu polymerase, .about.1 copy/μl B. subtilis genomic DNA(estimated by serial dilution) (20 μl reaction volume). Optimal primerspairs were J1+K2, and then J1C+K2C. In general a homopolymeric Cytosinestretch appeared to make nucleoprotein filaments more active, and aGuanosine stretch less active.

FIG. 48 shows the effects of betaine on RPA reactions

RPA reactions were established in which an increasing concentration ofbetaine was present in each reaction. Betaine concentration of 0.5M, orgreater, in this experiment reduced the degree of amplification. At0.75M the expected product band was still clearly visible, however themost of the smear present in some samples had been prevented. We believethat the reduction in smear far outweighs the reduction in productconsistent with the possibility that levels of betaine can be employedthat improve signal to noise ratios. Amplification conditions were: 50mM Tris.Cl pH 8.3, 90 mM Magnesium acetate, 2 mM DTT, 80 mM Potassiumacetate, 200 μM dNTPs, 600 ng/μl gp32, 200 ng/μl uvsX, 60 ng/μl uvsY,300 nM of primers ApoB4 and Apo300 (SEG IDs 20 and 21), 40 ng/μl Bsupolymerase, 2.5 copies/μl genomic DNA, 120 minutes at 37′C.

FIG. 49 shows combination strategies involving oligonucleotides withdifferent nucleoprotein activities

(A) Single reaction nesting strategies are outlined. An outer pair ofactive nucleoprotein filaments rapidly amplifies DNA, although theseprimers may also be fairly noisy. The outer primer pair may bemaintained at relatively low concentration, low enough to be unable toachieve gel-detectable levels of product. The inner oligonucleotides areless active, but much cleaner. These inner oligonucleotides aremaintained at high levels. Activity may be tuned by altering length,composition, and backbone character as detailed in this disclosure. Theouter primers quickly enrich the target in the sample, for examplethrough a million-fold amplification. The inner primers, being slow butclean, generate little/no noise, but are less sensitive. The enrichmentof target by the outer primers is sufficient to permit robust productaccumulation by action of the slow inner primers in an appropriatetimeframe. (B) Amplification schemes are shown in which an active primeris combined with a less active primer. While the active primer mayengage in noise at low target concentrations, the slower oligonucleotidedoes not engage in artifacts, and so if association of the primers istested at the end of the reaction this can cleanly determine whethertarget was presence or absent in the sample.

FIG. 50 describes several detection protocols. Three simple strategiesare shown schematically by which amplification of a target might beassessed without a need for gel electrophoresis. In all cases a primeris utilized that can be attached to a solid phase to permit itsseparation from bulk reactants, and which can be washed. In the figure abiotin residue on one of the primers represents this immobilizableprimer, however other attachment chemistries could be used. (1) Oneprimer is immobilizable, and the other contains a label of somedescription, for example an enzyme. (2) One of the amplification primersis labeled in some way, and the immobilizable primer is a third primer,which recognizes sequences within the main amplicons. This third primermay be present in the reaction environment during the mainamplification, but may be for example designed to be quiet (e.g. bybeing short), or could be added only at the end of the reaction. (3) Athird primer is employed. In this case however the immobilizable probeis related to one of the amplification primers, but contains additional5′ residues that permit it to stably capture amplicons at the end of thereaction, as polymerase extension of the 3′-end of the complement in theamplicon (i.e. ‘backfire’ synthesis) creates a stable duplex not subjectto branch migration if reaction proteins are washed away.

FIG. 51 shows a third probe enrichment strategy: Bead capture Part I.Schematic of experimental strategy in which a third probe enriches bonafide products derived from the target

(A) Schematic representation of the relationship between primers. A PstIrestriction enzyme site is indicated, located in the middle of NEST3-28(GGATGAATAAGCTGCAGCTGATTAAAGG—SEQ ID 76) and NEST3-26(ATGAATAAGCTGCAGCTGATTAAAGG—SEQ ID 77) primers which are homologous toan amplified sequence generated by primer J1 and K2 (SEQ IDs 66 and 69respectively above). (B) Illustration of the immobilization of a5′-biotinylated version of N3-26 which is bound to streptavidin-coatedmagnetic particles. (C) Scheme for capture experiment. An amplificationreaction is established in which oligonucleotides J1 and K2 are combinedin solution phase with the N3-26 primer immobilized on a bead. Thereaction is incubated for 90 minutes, and every 20 minutes the beads aredispersed by flicking (they settle only very slowly in the 5% PEGreaction solution). At the end of the incubation period the beads areconcentrated using a magnet, and the supernatant is removed foranalysis. The beads are washed twice quickly with water, and thenincubated for 30 minutes at 37° C. with excess PstI enzyme inappropriate buffer. Once again the supernatant is removed and analysed.

FIG. 52 shows a third probe enrichment strategy: Bead capture II.Experimental results demonstrating that a low activity nucleoproteinfilament immobilized on a solid support can participate as a thirdprimer and separate target amplicons from primer noise.

(A) Schematic representation of the relationship between primers. A PstIrestriction enzyme site is indicated, located in the middle of N3-28 andN3-26 primers. (B) Amplification of B. subtilis DNA has been performedin the presence (lower panel) or absence (upper panel) of target DNA at2 copies/μl. Lane 1 contains size ladder. The primers J1 and K2efficiently amplify their target in the presence of few copies of targetgenomic DNA (Lower panel, lane 2), however in the absence of target inthe sample generate a smear as evidenced in the upper panel lane 2.Oligonucleotides N3-28 and N3-26 are homologous to sequences slightly3′, and the same sense, to J1. N3-28 is a 28-mer, and N3-26 is a 26-merlacking the two 5′-most bases from N3-28. Co-incubation of N3-28 orN3-26 with J1 and K2 primers results in the generation of two mainproducts corresponding to the J1/K2 product, and the expected N3-28/K2or N3-26/K2 product (Lower panel, lanes 3 and 4). When N3-28 isincubated alone it generates some smear, but N3-26 incubated alone doesnot. We deduce that N3-26 is a ‘quiet’ olignucleotide and may relysignificantly on hybridization to displaced strands from the elongationof more active primers in order to generate products. (C) The results ofan experiment carried out as detailed schematically in FIG. 51, andusing start target density of 30 copies/μl. Lane 1, size marker. Lane 2and 3, products of an entirely liquid phase reaction (no immobilizationof N3-26) performed without (lane 2) or with (lane 3) target. This iseffectively equivalent to lanes 4 of the upper and lower panels of (A).Lanes 4 and 5 contain the first supernatant removed after theamplification incubation, without and with target respectively. Asexpected there is product in the target containing reaction, and thesmaller product derived from use of the N3-26 primer (immobilized on thebead) is not seen. Also there is a smear, as expected, in the targetlesssample. Lanes 6 and 7 contain the supernatant following PstI digestionof the washed beads. Interestingly the smear in the non-target sample isnot present, however in the target sample there is DNA. The productsreleased from the beads appear to comprise the expected N3-26/K2product, slightly faster mobility than in lane 3 due to clipping byPstI, as well uncut J1/K2 product that has co-purified. It is perhapsunsurprising that some J1/K2 product co-purifies because hybrids maywell have formed between such products and the bead-immobilized productsand oligonucleotides. It is, however, surprising that this material hasnot apparently been cut by PstI enzyme. The reason for this strangeobservation is not known, but could indicate that these products werepresent in hybrids that were resistant to digestion, or lead to nickingof only one strand of these products.

FIG. 53 shows that Trehalose stabilizes lyophilizates to permit allcomponents except buffered sample to remain active for at least 10 daysat room temperature.

RPA reactions were assembled using primers specific for the humanApolipoprotein B locus. In general final reaction composition was 55 mMTris.Cl pH 8.4, 1 mM DTT, 80 mM Potassium acetate, 5% Carbowax 20M, 200μM dNTPS, 3 mM ATP, 20 mM Phosphocreatine, 100 ng/μl creatine kinase,300 nM ApoB4 and Apo300 oligonucleotide (SEQ ID 20 and 21 respectively),600 ng/μl gp32, 200 ng/μl uvsX, 60 ng/μl uvsY, and 17 ng/μl Bsupolymerase, ˜160copies/μl Human genomic DNA. However, the 55 mM Tris.ClpH 8.4 and 80 mM Potassium acetate were not included in thelyophilizate, but added back with the DNA sample during reconstitution.Furthermore certain components were also omitted from the lyophilizatesdetailed as follows. Condition A, no trehalose used, Carbowax added withsample DNA (not in lyophilizate), polymerase added with sample DNA.Condition B, 50 mM Trehalose included in lyophilizate, Carbowax addedwith sample DNA, polymerase added with sample DNA. Condition C, 50 mMtrehalose in lyophilizate, polymerase added with sample. Condition D, 50mM trehalose in lyophilizate, only buffered sample DNA added forreconstitution. After 3, 6, or 10 days storage on the bench at roomtemperature, and with no special storage conditions (e.g. use ofdessicants etc.) the activity of the lyophilizates were tested by addingthe missing reagents to the dry pellet in a final volume of 50 μl.Reaction condition D in each case lacked only the sample DNA in reactionbuffer (55 mM Tris.Cl and 80 mM Potassium acetate). Only if trehalosewas present in the lyophilizate were reactions stable, but in thosecases the reactions remained stable for at least 10 days. Longer periodswere not investigated due to running out of test material, but may bepossible.

Example 14 RPA Reactions can be Monitored in Real Time to AllowAssessment of Start Target Copy Number

FIG. 54 shows how SYBR gold and SYBR green fluorescent dyes wereassessed for their compatibilities in RPA reactions, and theirreaction-monitoring properties. (A)

Arrangement of the primers ApoB4 and Apo300 (SEQ IDs 20 and 21respectively) which flank a fragment of the human Apolipoprotein Blocus. (B) SYBR gold can be included in RPA reactions at dilutions of1:50,000 or greater without complete inhibition of the reaction.Reactions were composed of 50 mM Tris pH 8.4, 80 mM Potassium acetate,200 μM dATP, dCTP, dGTP, and dTTP, 50 ng/μl creatine kinase, 1.5 mM ATP,10 mM Magnesium acetate, 2 mM DTT, 30 mM phosphocreatine, 300 nM ApoB4primer, 300 nM ApoB300 primer, 5% Carbowax 20M, 360 ng/μl gp32, 86 ng/μluvsX, 15 ng/μl uvsY, 35 ng/μl Bsu polymerase. SYBR gold at the indicateddilution was included in the reaction. (C)RPA reactions were establishedin a 96-well plate, each with a final volume of 50 μl. A mastermix ofcomponents lacking only the target DNA and the SYBR gold dye wasestablished on ice. Five different dilutions of SYBR gold were tested,with final dilutions from stock of 1:50,000, 1:60,000, 1:70,000,1:80,000, and 1:100,000. For each dilution starting target copydensities of 2 and 20 copies per microliter were investigated (humangenomic DNA diluted in TE to the expected copy density). Once reactionshad been thoroughly mixed on ice, the plate was transferred to thepre-warmed plate (37° C.) of a BIO-TEK FLX 800 fluorescent microplatereader. Readings were collected every 1 minute, and 300 ng of humangenomic DNA diluted in 50 μl of water was used as a sensitivity standardfor the device. Data was transferred to Microsoft Excel and relativefluorescence (arbitrary) was plotted against incubation time for eachreaction. (D) An experiment was established as in (C) except that SYBRgreen rather than SYBR gold was assayed.

FIG. 55 shows that real time RPA demonstrates quantitative behaviourover four order of magnitude using SYBR green. FIG. 55 (A) Arrangementof primers used in this analysis. (B) RPA reactions were established in96-well microwell plates, with a final volume of 50 μl per reactionvolume, using primers specific or the B. subtilis SpoOB locus J1 and K2(J1-5′-ACGGCATTAACAAACGAACTGATTCATCTGCTTGG-3′ SEQ ID 66,K2-5′-CCTTAATTTCTCCGAGAACTTCATATTCAAGCGTC-3′ SEQ ID 69). A definednumber of copies of Bacillus subtilis genomic DNA was pipetted intowells in to the 96-well plate, and stored on ice. A reaction mastermixwas mixed on ice, and then aliquoted in the wells containing DNA andmixed well. The reactions were then transferred to the pre-warmed plateof a BIO-TEK FLX 800 fluorescent microplate reader (set at 38° C.). Thereaction composition was 50 mM Tris pH 8.4, 80 mM Potassium acetate, 10mM Magnesium acetate, 2 mM DTT, 200 μM dATP, dCTP, dGTP, and dTTP, 50ng/μl creatine kinase, 1.5 mM ATP, 30 mM phosphocreatine, 300 nM BsJ1primer, 300 nM BsK2 primer, 5% Carbowax 20M, 360 ng/μl gp32, 86 ng/μluvsX, 15 ng/μl uvsY, 35 ng/μl Bsu polymerase, and 1:50,000 dilution ofthe DMSO stock of SYBR green from molecular probes. The results of twoequivalent experiments is shown in (B) and (C). After reactions hadfinished 7 μl of the reactions was diluted with 1× sucrose loadingbuffer and separated on a 2% agarose gel (D, E). Note that when thehighest concentration of target was used fluorescence became detectableearliest as expected, but failed to show an enduring exponential rise.This may arise due to consumption of reaction reagents (gp32 was used atlower than typical concentrations in this experiment), or to aninhibitor build-up, or otherwise. Later experiments suggest this mayarise due to gp32/uvsX undertitration. See below. Also, there someevidence of product contamination in (B) suggested by the faint presenceof the expected band in the endpoint analysis gel (D).

FIG. 56 shows that RPA reactions can kinetically assess of B. subtilisgenomic DNA copy number of at least 5 orders of magnitude. RPA reactionswas established using Bacillus subtilis primers J1 and K2 as in FIG. 55.However the quantity of protein reagents was increased as follows: gp32was used at 600 ng/μl, uvsX was used at 140 ng/μl, uvsY was used at 35ng/μl, and ATP was at 3 mM. Reaction volumes were increased to 100 μlfinal volume, and a greater range of genomic DNA concentrations was used(from 0.1 to 100,000 copies/μl). No early trailing of higher copy numbersamples was observed, and we believe this reflects that earlierexperiments had somewhat undertitrated gp32 and uvsX. After reactionshad finished 7 μl of the reactions was diluted with 1× sucrose loadingbuffer and separated on a 2% agarose gel.

FIG. 57 shows that real time RPA demonstrates quantitative response tovariation in human genomic DNA copy number. FIG. 57 (A) Arrangement ofApoB4 and Apo300 primers which amplify a human DNA sequence.(ApoB4--5′-CAGTGTATCTGGAAAGCCTACAGGACACCAAAA3′ SED ID 20,Apo300--5′-TGCTTTCATACGTTTAGCCCAATCTTGGATAG3′ SEQ ID 21). (B) RPAreactions were established in 96-well microwell plates, with a finalvolume of 100 μl per reaction volume. A defined number of copies ofhuman genomic DNA was pipetted into wells in to the 96-well plate, andstored on ice. A reaction mastermix was mixed on ice, and then aliquotedin the wells containing DNA and mixed well. The reactions were thentransferred to the pre-warmed plate of BIOTEK FLX 800 fluorescentmicroplate reader. The reaction composition was 50 mM Tris pH 8.4, 80 mMPotassium acetate, 10 mM Magnesium acetate, 2 mM DTT, 200 μM dATP, dCTP,dGTP, and dTTP, 50 ng/μl creatine kinase, 1.5 mM ATP, 30 mMphosphocreatine, 300 nM ApoB4 primer, 300 nM Apo300 primer, 5% Carbowax20M, 360 ng/μl gp32, 86 ng/μl uvsX, 15 ng/μl uvsY, 35 ng/μl Bsupolymerase, and 1:50,000 dilution of the DMSO stock of SYBR green frommolecular probes. Note that, as in Example 2 (C), the highestconcentration of target generated a curve which trailed off unexpectedlyearly. As with FIG. 55 these experiments were performed under conditionsof uvsX, uvsY, gp32, ATP and dNTP concentrations that may requirefinessing to ensure robust detectable exponential phase at higher targetconcentrations. This is supported by (C), in which a similar experimentwas performed to that in (B), except that concentrations of certainreagents were as follows: gp32 was used at 600 ng/μl, uvsX was used at140 ng/μl, uvsY was used at 35 ng/μl, ATP was at 3 mM. After reactionshad finished 7 μl of the reactions was diluted with 1× sucrose loadingbuffer and separated on a 2% agarose gel.

Example 15 Asymmetric Primer RPA Using a 3′LNA-Capped Primer

As an example of amplification of a target DNA using the asymmetricprimer RPA method, standard RPA conditions are employed with twodifferences. First, one primer is a 3′LNA-capped primer. Secondly, anadditional polymerase, phi29, is used, which is unable to initiatesynthesis from recombinase intermediates.

Thus amplification conditions are as follows:

-   -   50 mM Tris.Cl pH 7.85    -   10 mM Magnesium acetate    -   2 mM Dithiothreitol    -   80 mM Potassium acetate    -   5% PEG compound (Carbowax 20M)    -   15 mM Phosphocreatine    -   3 mM ATP    -   200 μM dNTPs    -   12 ng/μl creatine kinase    -   0.3 μM oligonucleotide 1, 3′LNA-capped    -   0.3 μM oligonucleotide 2    -   350 ng/μl T4 gp32    -   100 ng/μl T4 uvsX    -   20 ng/μl T4 uvsY    -   20 ng/μl Bsu polymerase    -   20 ng/μl phi29 polymerase (exo-, or exo-attenuated)    -   Incubation at 33-40° C. for 30 minutes to 2 hours

Example 16 Initiation of Real-Time RPA with Deprotection of Caged ATP

As an example of the use of ATP pulsed RPA to quantify input templateDNA reactions are assembled as standard RPA reactions with severaldifferences. Firstly, caged-ATP is used instead of ATP. Secondly, adouble-stranded DNA detection reagent, SYBR green, is used to quantifyRPA products. Primers are designed such that short (<200 bp) reactionproducts are generated, to ensure complete synthesis with each pulse ofATP.

Thus amplification conditions are as follows:

-   -   50 mM Tris.Cl pH 7.85    -   10 mM Magnesium acetate    -   2 mM Dithiothreitol    -   80 mM Potassium acetate    -   5% PEG compound (Carbowax 20M)    -   3 mM caged-ATP    -   1:50,000 dilution of SYBR-green I commercial stock (Molecular        Probes)    -   2001M dNTPs    -   0.3 μM oligonucleotide 1    -   0.3 μM oligonucleotide 2    -   600 ng/μl T4 gp32    -   150 ng/μl T4 uvsX    -   25 ng/μl T4 uvsY    -   20 ng/μl Bsu polymerase    -   Incubation at 33-40° C.

Prior to each cycle of ATP uncaging, SYBR-green I fluorescence (494 nmExcitation Max-521 nm Emission Max) is measured in the sample. Thedetected fluorescence will be the basis of quantification. Every minutea short pulse of 365 nm light is applied to uncage ATP, leading to aburst of recombinase activity. Fluorescence measured at each cycle isthen compared to an input template standard and the quantity of inputsample template can be determined.

Example 17 Describes the Use of Caged ATP to Control the Initiation ofRPA in a Polony Analysis

As an example of RPA-based polony analysis polyacylamide gels aregenerated on microscope slides as described by Church and colleagues[Mitra R, Church G (1999) In situ localized amplification and contactreplication of many individual DNA molecules. Nucl Acids Res 27(24):e34i-vi.]. In this analysis distinct polymorphic products are detectedafter an initial amplification by single-base extension usingfluorescent nucleotides and polymorphic-target specific primers. Onedifference between this configuration and normal polony analysis is thatit may not be necessary to use the 5′acrydite modified primers, as RPAperformed at a low constant temperature and for significantly shortertimes (normally PCR-based polony reactions would take 3-7 hours). Inthis example, both 5′acrydite modified primers as well as normal primersare used.

In place of the PCR “diffuse-in” mix, an RPA “diffuse-in” mix is usedand comprises the following:

-   -   50 mM Tris.Cl pH 7.85        10 mM Magnesium acetate

2 mM Dithiothreitol

80 mM Potassium acetate5% PEG compound (Carbowax 20M)15 mM Phosphocreatine (included depending upon configuration)200 μM dNTPs* 3 mM caged-ATP (included depending upon configuration)12 ng/μl creatine kinase (included depending upon configuration)0.3 μM oligonucleotide 10.3 μM oligonucleotide 2350 ng/μl T4 gp32100 ng/μl T4 uvsX20 ng/μl T4 uvsY20 ng/μl Bsu polymerase

The reaction is then initiated either by diffusing in ATP, or activatingcaged-ATP included in the original “diffuse-in” mix. Polonies aregenerated after incubation at 33-40° C. for 30 minutes to 2 hours

After amplification, a new polymorphic-target primer is diffused inalong with fluorescently labelled nucleotides, and the reaction isre-started with an additional pulse of ATP, either applied manually, orby photolysis. Incorporated fluorescence can then be quantified usingfluorescence microscopy.

Example 18 Non Gel-Based Determination of Polymorphic Repeat Number inan Amplicon Using Recombinase-Mediated Hybrid Formation BetweenSingle-Stranded Amplified DNA and Duplex Probes

In this example an STR is amplified by flanking oligonucleotide primers.The STR is the marker known as TPOX and commonly used in both US andEuropean standard STR marker sets used for forensic analysis. The markeris amplified by flanking primers with the sequence for primer 1 of5′-ACTGGCACAGAACAGGCACTTAGGGAACCC-3′ (SEQ ID NO: 44), and primer 25′-GGAGGAACTGGGAACCACACAGGTTAATTA-3′ (SEQ ID NO: 45). The marker has arepeat of 4 nucleotides, AATG, which vary between 5 and 14 repeats.

Amplification conditions are as follows:

-   -   50 mM Tris.Cl pH 7.85    -   10 mM Magnesium acetate    -   2 mM Dithiothreitol    -   80 mM Potassium acetate    -   5% PEG compound (Carbowax 20M)    -   15 mM Phosphocreatine    -   3 mM ATP    -   200 μM dNTPs    -   12 ng/μl creatine kinase    -   0.3 μM oligonucleotide 1,5′-fluorescein-labeled    -   0.05 μM oligonucleotide 2    -   600 ng/μl T4 gp32    -   150 ng/μl T4 uvsX    -   35 ng/μl T4 uvsY    -   20 ng/μl Bsu polymerase    -   Incubation at 33-40° C. for 30 minutes to 2 hours

The amplification reaction generates a quantity of single-stranded DNAdue to primer imbalance, and this single-stranded DNA is fluorescentlylabeled. At the end of the amplification reaction the reaction mixtureis contacted directly to an array of spatially immobilizeddouble-stranded probes. Each probe corresponds to one of the knownpolymorphic forms of the DNA under study, and is the same length as theamplicon that would be made if this form exists in the sample.

Recombinase loads onto the amplified single-stranded sample DNA to formhomology-searching filaments. These filaments will be able to target thedouble-stranded probes immobilized on the array support surface. When arecombination event occurs between the recombinase-coated amplificationproduct and the probes it will lead to the amplification productbecoming productively Watson-Crick base-paired with its complement inthe probe, while the originally paired equivalent strand is displacedand freed into solution. Ideally completion of such recombination eventswill be inhibited if the event initiates between a sample DNA and animperfectly matched probe leading to enrichment of only perfect hybrids.

The reaction is dynamic such that hybrids formed between probe andsample may themselves become targets for other targeting DNAs. Thisdynamic behavior coupled to a lower efficiency of completion ofrecombination of imperfectly formed hybrids should lead to an enrichmentof the perfect hybrids in the population of probe/sample mixtures. Thisis further enhanced by the addition of either a helicase such as E. coliPriA (2-20 ng/μl), E. coli DnaB (5-50 ng/μl), E. coli RuvAB (5-50ng/μl), T4 phage dda helicase (5-50 ng/μl), T4 phage gp41 (5-50 ng/μl)or an appropriate nuclease. These agents utilize, or enhance,disruptions in the helix, which exist due to mismatches and lead to suchimperfect hybrids being less stable than perfect hybrids.

After a suitable incubation period, such as 10-30 minutes at 28-40° C.,the reaction is terminated if necessary, for example by the addition ofEDTA or chaotropic agents such as urea or guanidine hydrochloride,although no such termination may be necessary. Productive interactionsbetween probe and sample are then measured by exposing the array tolight of an appropriate wavelength, and recording fluorescence usingappropriate filters and camera.

Example 19 Shows a Non Gel-Based Determination of Polymorphic RepeatNumber in an Amplicon Using Recombinase-Mediated Hybrid FormationBetween Duplex Amplified DNA and Single-Stranded Probes

As above this example would involve amplification of the TPOX STR markerused in forensics. The amplification conditions would be the same as forExample 18 except that the ratio of amplification primers would be 1:1,with both used at a concentration of 0.3 μM. The amplification reactionis then contacted to an array of single-stranded probes corresponding tothe known possible polymorphic forms of the DNA under study. With thisformat the recombinase loads preferentially onto the probes which thensearch and form hybrids with the double-stranded sample DNAs. As inExample 18 hybrids will ideally become enriched if they are perfect, andsimilarly the inclusion of agents such as helicases (e.g. PriA, recG,DnaB, RuvAB, gp41, or dda) or appropriate nucleases can accelerate andimprove this enrichment. After a suitable incubation period, with orwithout arrest of the reaction as necessary, interactions are visualizedas described in Example 18.

Example 20 Shows a Non Gel-Based Determination of Polymorphic RepeatNumber in an Amplicon Using Recombinase-Mediated Hybrid Formation andNuclease Processing of Heterologies to Release a Label from anImmobilization Point

In this example the amplification reaction and hybrid formation betweenprobe and sample are performed as described in Example 1, except thatthe amplification primers need not contain a label. The probe instead ofthe sample is labeled at one end. After an appropriate incubationperiod, as described for Examples 18 and 19, arrays are treated with anappropriate nuclease, which will cleave imperfect DNA duplexes. As suchcuts are made in the backbone of imperfect hybrids and the label is thusfreed from an immobilization point. Additionally a DNA polymerase may beincluded such that if a nick is introduced by the nuclease, and thepolymerase is strand displacing then it acts to synthesize in astrand-displacing manner and thus remove the labeled strand accordingly.The array can be then quantified as described in Example 18.

Example 21 Shows a Non Gel-Based Determination of Polymorphic RepeatNumber in an Amplicon Using Recombinase-Mediated Hybrid Formation andNuclease Processing of Heterologies Followed by DNA Synthesis Labeling

In this example amplification and hybrid formation are as for eitherExample 18 or Example 19 except that the amplification primers are notnecessarily labeled. During the hybrid formation phase a nuclease isincluded that nicks at bubbles or helix disturbances and the inclusionin the reaction of labeled nucleotides and a strand displacingpolymerase leads to imperfect hybrids being processed to a perfecthybrid form which includes modified labeled bases that can then bevisualized.

Example 22 Shows the Use of a Helicase to Disrupt Imperfect Hybrids in aDynamic Recombination System Environment

In this example amplification and hybrid formation is done as for eitherExample 18 or Example 19 except that the amplification primers are notnecessarily labeled. The probe or sample DNA should however contain alabel. The probe or sample DNA is immobilized at one end to a solidsupport or bead through a high affinity non-covalent interaction such asa biotin-streptavidin interaction. During the hybrid-forming phase ahelicase such as the T4 dda helicase, or combination of helicases suchas PriA and dda helicases, or other mixtures are include to targetimperfect hybrids and unwind them, effectively accelerating thedissociation of high affinity non-covalent interaction by physicaldisruption. After a suitable incubation period the association betweenthe labeled nucleic acid and the solid support is measured positivesignal indicating that perfect hybrids have formed.

FIG. 58 shows in principal how imperfect hybrids will possess bubbleswhich permit enhanced overall duplex disruption in a dynamicrecombination environment

A double-stranded amplicon shown in A containing 8 repeats of a shorttandem repeat and flanked by unique sequences used for amplification istargeted in B by single-stranded probe oligonucleotides containingvarying numbers of the repeat unit. The hybridization is driven byrecombinase (not shown here). In C the resultant double-stranded hybridsare shown, some of which contain bubbles caused by non-identical repeatnumbers between hybrids. These bubbles are larger as the number ofrepeats different grows in number, and thus such hybrids become bettertargets for recombinase, or other DNA processing enzymes.

FIG. 59 shows the possible outcome of a hybridization reaction asdescribed in Example 18, of FIG. 58, is shown. A sample DNA containing 8repeats is incubated with an array of probes corresponding to differentrepeat numbers. Over time hybrids are enriched for perfect repeatnumbers, and less so but to a small extent those that differ only by oneor two repeats.

FIG. 60 shows how easily RPA reactions can be performed to assess thepresence of specific RNA species by including an enzyme capable ofreverse transcription in the reaction.

Defined copy numbers of viral MS2 RNA (from a commercial source) wereincubated in RPA reactions which included reverse transcriptase asindicated. Two primers were employed which recognised the MS2 sequenceas indicated, more specifically these primers were:

MS2up2 SEQ ID 78 TTCCGACTGCGAGCTTATTGTTAAGGCAATG MS2down2 SEQ ID 79CTTAAGTAAGCAATTGCTGTAAAGTCGTCAC

Reaction conditions were standard except for increased concentration ofdNTPs (500 μM), an increase in DTT, the inclusion of RNase inhibitor,and the inclusion of reverse transcriptase where indicated. Conditionswere:

-   -   50 mM Tris.Cl pH 8.5    -   10 mM Magnesium acetate    -   10 mM Dithiothreitol    -   80 mM Potassium acetate    -   5% PEG compound (Carbowax 20M)    -   15 mM Phosphocreatine    -   3 mM ATP    -   500 μM dNTPs    -   100 ng/μl creatine kinase    -   0.3 μM MS2UP    -   0.3 μM MS2DOWN    -   800 ng/μl T4 gp32    -   120 ng/μl T4 uvsX    -   30 ng/μl T4 uvsY    -   70 ng/11 Bsu polymerase    -   0.13 Units/μl RNase inhibitor    -   MuMLV (RNase H containing) reverse transcriptase 10 units/μl        (Promega)

Reactions products were phenol extracted, precipitated and separated ona non-denaturing acrylamide gel before staining with SYBR-gold. RNAconcentrations as low as 100 copies per microliter can be readilydetected in this format without further optimization.

FIG. 61 shows that dUTP can be used to partially or completely replacedTTP in RPA reactions, thus offering a strategy to control carry-overcontamination.

RPA reactions were established using the following conditions:

-   -   50 mM Tris.Cl pH 8.5    -   10 mM Magnesium acetate    -   2 mM Dithiothreitol    -   100 mM Potassium acetate    -   5% PEG compound (Carbowax 20M)    -   15 mM Phosphocreatine    -   3 mM ATP    -   200 μM dNTPs (dA, dC & dG)    -   100 ng/μl creatine kinase    -   0.3 μM J1 primer for B. subtilis DNA (SEQ ID 66)    -   0.3 μM K2 primer for B. subtilis DNA (SEQ ID 69)    -   630 ng/μl T4 gp32    -   140 ng/μl T4 uvsX    -   35 ng/μl T4 uvsY    -   20 ng/μl Bsu polymerase    -   100 copies/μl B. subtilis genomic DNA

The product of reactions was separated on a 2% agarose gel and stainedwith ethidium bromide. As indicated in the left-most panels, dUTP ordTTP was employed in different reactions. More specifically the upperleft-most lane contained 200 μM dTTP, and was therefore typical of astandard RPA reaction. The following six lanes also contained 200 μMdTTP, but in this case dUTP was also present at the indicatedconcentrations. The last six lanes of the lower left-most panel containthe products of RPA reactions in which no dTTP was included, and theindicated concentration (50-800 μM dUTP) of dUTP was used. In all casesamplification of the expected fragment occurred, but notably thepresence of dUTP decreased the quantity of amplicon generated, andinhibited the ‘doubling-up’ phenomenon often seen in which snap-backsynthesis has occurred.

The right-most panel indicates the results of using as starting materialproducts generated from the first reactions in the left-most panelfollowing processing as indicated in the schematic. More specificallythe reactions shown as A, B, C, D and E in the left-most panels wereprocessed as follows. Based on staining intensity and estimation of DNAcontent, an estimated 10⁶ molecules of each product were incubated for20 minutes with 1 μl of a commercial preparation (Roche) of heat-labiledUTP-deglycosylase, a similar amount was left untreated, and thenfinally all samples were heated to 94° C. for 10 minutes. This enzymewas quality-controlled by the manufacturer to effectively combatcarry-over contamination from 10⁵ molecules, thus we imposed a stringentchallenge in this case. The samples were then used to seed an RPAreaction configured under normal RPA conditions with 200 μM of eachdATP, dCTP, dGTP, and dTTP (see conditions above). As indicated samplesuntreated with dUTP-deglycosylase were excellent start templates, whilethose that had been treated were extremely poor unless no dUTP had beeninitially employed. In particular even the mixed blends were effective.

FIG. 62 shows how one might combat carry-over contamination in the RPAsystem.

The capacity to employ dUTP in RPA reactions could be used to preventcarry-over contamination according to the depicted scheme. RPA reactionswould be performed with pure dUTP, or mixes of dTTP and dUTP as shown inFIG. 61. Prior to, or at the initiation of an RPA reaction, E. coliUracil deglycosylase (UDG) or similar would be incubated with the sampleand would attack carry-over material. Then after several minutes, or atthe start of the reaction, an UDG inhibitor would be added. A specificinhibitor of E. coli UDG is known as is referred to as Uracil GygosylaseInhibitor (UGI) and is a 9.5 kD peptide from Bacillus subtilis, and iscommercially available.

FIG. 63 shows how a simple integrated disposable system can beconfigured to permit rapid point-of-use testing for the presence ofspecific DNA sequences in a sample. In (A) a schematic description isgiven of a disposable RPA reaction/lateral flow strip that might beplaced into a cheap heater than can deliver roughly 30-39° C. in thevicinity of a reaction pouch containing dried RPA reagents. It isassumed that processed/lysed sample would be used to rehydrate thecontents of the pouch in an appropriate volume, and then the pouch wouldbe incubated at appropriate temperature for 15-60 minutes, as necessary.Subsequently part or all of the sample would be transferred to thesample pad of a lateral flow strip, configured in a manner that only ifthe correct amplicons have formed will a visible line form at a specificposition on the strip. In (B) is shown the arrangement of twooligonucleotide primers used for an RPA reaction performed on male andfemale DNA respectively. The oligonucleotides SRY3 and SRY4 were used,and one contained a 5′-fluorescein moiety, and the other a 5′-biotinmoiety. At the end of the reaction 1/500 of a 50 μl reaction was mixedwith ‘running buffer’ (Milenia, Germany) and applied to acommercially-available lateral flow strip (Milenia, Germany) such thatonly products in which the fluorescein and biotin were co-associated(i.e. in an amplification product) would lead to the accumulation ofvisible gold particles on the detection line. In (C) the result of thisexperiment is shown. Only the reaction performed in male DNA generatesthe signal line, and the efficacy of the amplification reaction wasseparately validated on agarose gels (data not shown).

FIG. 64 shows that RPA reactions are not inhibited by the presence ofmaterials in blood providing appropriate ratios of reagents areemployed.

The ability to amplify a genomic DNA fragment from whole and lysed bloodadded directly to RPA reactions was investigated. RPA reactions wereconfigured as follows:

-   -   50 mM Tris.Cl pH 8.5    -   10 mM Magnesium acetate    -   2 mM Dithiothreitol    -   80 mM Potassium acetate    -   5% PEG compound (Carbowax 20M)    -   25 mM Phosphocreatine    -   3 mM ATP    -   200 μM dNTPs    -   100 ng/μl creatine kinase    -   0.3 μM oligonucleotide ApoB4 (SEQ ID 20)    -   0.3 μM oligonucleotide ApoB300 (SEQ ID 21)    -   420 ng/μl T4 gp32    -   140 ng/μl T4 uvsX    -   35 ng/μl T4 uvsY    -   20 ng/μl Bsu polymerase

In (A), either 1 μl of water or 1 μl of fresh whole human blood wasadded to an RPA reaction. Products were separated on a 2% agarose gel,and an arrow indicates the position of the expected amplicon.Non-specific ‘primer’ artifacts were present in water control, and inthe sample, and presumably arise as a consequence of absent or very lowcopies of target. We deduce that very few copies of genomic DNA wereavailable for amplification in the blood sample amplification. In (B)samples were analysed in which 1 μl of blood had first been mixed witheither 1 μl, 2 μl, 3 μl, 4 μl, or 5 μl of a lysis solution comprising 10mM|Tris, 1 mM EDTA, 120 mM NaOH, 0.1% SDS (as indicated). In each case 1μl of the respective lysate was used to start an RPA reaction (thusthose lysed with more buffer contained less blood sample peramplification). Also shown are samples from the same experiment in whichwater only (lane 1), or purified DNA equivalent to 500 copies of target(Promega)(lane 7) were used to seed the amplification reaction. When 4μl or 5 μl of lysis buffer were used, lysis of the red blood cells wasclearly visible and the solution became viscous. We note that thesesamples became excellent start materials for RPA reactions, presumablyreleasing most of the DNA as accessible templates.

FIG. 65 shows how real-time monitoring of RPA reactions can be employedto optimise reactions.

In this case RPA reactions were established using the primer pair J1 andK2 mentioned above, and template consisting of Bacillus subtilis genomicDNA (start density 1 copy per microliter). Either Potassium acetateconcentration, or PEG compound concentration, was varied.

Other conditions were, unless specified otherwise:

-   -   50 mM Tris.Cl pH 8.5    -   10 mM Magnesium acetate    -   2 mM Dithiothreitol    -   80 mM Potassium acetate (or as indicated)    -   5% PEG compound (Carbowax 20M)(or as indicated)    -   25 mM Phosphocreatine    -   3 mM ATP    -   200 μM dNTPs    -   100 ng/μl creatine kinase    -   0.3 μM oligonucleotide J1 primer (SEQ ID 66)    -   0.3 μM oligonucleotide K2 (SEQ ID 69)    -   620 ng/μl T4 gp32    -   140 ng/μl T4 uvsX    -   35 ng/μl T4 uvsY    -   1:50,000 dilution form stock of SYBR green (Invitrogen)

20 ng/μl Bsu polymerase. At the end of the reaction a portion of thereactants were mixed with sucrose loading buffer and separated on anagarose gel, and then stained with ethidium bromide. We note that highsalt slows the reaction, but there is a broad range of saltconcentrations that work well. We also note that increasing PEGconcentrations from 4% to 9% resulted in ever faster reaction behaviour,however the appearance of products in the gel system was adverselyaffected by higher PEG concentrations. We surmise that the smearyappearance of high PEG concentration samples may arise from ongoingnetworks of DNA generated by very high recombination activity. Whilethis may be undesirable for gel analysis, it may be perfectly adequatefor real-time analysis using fluorescence.

FIG. 66 shows how RPA reactions are affected by Magnesium concentration.

Real-time RPA reactions were established as follows:

-   -   50 mM Tris.Cl pH 8.5    -   6, 10, or 16 mM Magnesium acetate as indicated    -   2 mM Dithiothreitol    -   80 mM Potassium acetate    -   5% PEG compound (Carbowax 20M)    -   25 mM Phosphocreatine    -   3 mM ATP    -   200 μM dNTPs    -   100 ng/μl creatine kinase    -   0.3 μM oligonucleotide J1 primer (SEQ ID 66)    -   0.3 μM oligonucleotide K2 primer (SEQ ID 69)    -   600 ng/μl T4 gp32    -   120 ng/μl T4 uvsX    -   30 ng/μl T4 uvsY    -   20 ng/μl Bsu polymerase    -   1:50,000 dilution from stock of SYBR green (Invitrogen)

The start density of target DNA, Bacillus subtilis genomic DNA, incopies per microliter is indicated. In the 6 mM and 16 mM Magnesiumacetate samples some carry-over contamination was evident when samplesat end-point were checked on agarose gels, and this may in part explainthe poor resolution between zero and 1 copy per microliter startdensity. Note that RPA is effective at all tested Magnesiumconcentrations, but is much faster as the Magnesium concentration rises.End-products from these higher Magnesium samples still appear of highquality (data not shown). This indicates that in order to achievemaximal reaction rates, Magnesium concentrations significantly higherthan 10 mM (used as a standard through most of this document) may beusefully employed.

FIG. 67 shows that different primer pairs/amplicons show differentamplification kinetics. Reaction time dependence varies betweenoligonucleotide primers, and analysis indicates a possible sequence-biasunderlying this phenomenon.

Two different timecourses were performed using different primer pairs.Conditions were not identical between these reactions, nevertheless thelevel of variation indicated is typical of the range of behaviours thatwe have observed between primer pairs in our hands and is included asjust one typical example of rates that are observed (CSF primers used at125 ng/μl UvsX, 45 ng/μl uvsY; Apo primers used at 200 ng/μl UvsX, 60ng/μl uvsY). Primer sequences are shown. Primers CSF5′(SEQ ID 32) andCSF3′ (SEQ ID 33)(targeting the human STR locus CSF1PO) are really anexceptional primer pair insofar as they demonstrated particularly rapidproduct accumulation kinetics, the fastest we have as yet determined.Primers ApoB600 (SEQ ID 46) and ApoB300rev (SEQ ID 47), targeting partof the human Apoliprotein B locus, demonstrate slower amplificationkinetics. In this experiment this latter pair generated a similar amountof material after 40 minutes as occurred after only 20 minutes with theCSF primers. Based on this, and other, data we conclude that typicaldoubling times vary, on the average, between about 30 seconds and 1minute for most primer pairs of 30-35 residues in RPA. CSF primers werealso notably dominant in multiplex experiments (data not shown),consistent with the notion that they were particularly efficient andfast. Composition analysis of the primers, comparing both CSF primerswith, in this case, the Apo primers used here, highlights that guanosinecontent is low in both CSF primers, while average in the Apo primers,and cytosine content is one third or more for both CSF primers, whileone quarter or less for the Apo primers. This suggested to us a possiblecomposition origin for rate variability. Reactions were performed undersimilar conditions apart from the aforementioned differences in UvsX andUvsY, and slightly variant dNTP concentrations:

-   -   50 mM Tris pH 8.4    -   80 mM Potassium acetate    -   10 mM Magnesium acetate    -   2 mM DTT    -   5% PEG compound (Carbowax-20M)    -   3 mM ATP    -   20 mM Phosphocreatine    -   100 ng/.quadrature.1 creatine kinase    -   600 ng/ml gp32    -   200.quadrature.M dNTPs for Apo primer, 1000M dNTPs for CSF        primers    -   300 nM each oligonucleotide

FIG. 68 shows that appended 5′ homopolymeric stretches influencereaction behaviour—cytosine increases activity and guanosine decreasesit.

Primers were as indicated. J1 and K2 (SEQ IDs 66 and 69) are homologousto part of the Bacillus subtilis SpoOB locus. Reaction conditions were:

-   -   50 mM Tris pH 8.4    -   80 mM Potassium acetate    -   10 mM Magnesium acetate    -   2 mM DTT    -   5% PEG compound (Carbowax-20M)    -   3 mM ATP    -   30 mM Phosphocreatine    -   100 ng/μl creatine kinase    -   420 ng/μl gp32    -   140 ng/μl UvsX    -   35 ng/μl UvsY    -   2000M dNTPs    -   300 nM each oligonucleotide    -   35 ng/μl Bsu polymerase    -   25 copies/μl B. subtilis genomic DNA    -   1:50,000 dilution from stock of SYBR green (Molecular probes)    -   Reaction volumes 50 μl, 37° C.

Primers J1(C), K(C), J1(G), and K2(G) were equivalent to J1 and K2, butpossessing appended stretches of cytosine or guanosine, as indicated.Reactions were established on ice in a microtitre plate, and thentransferred to the heated stage of a fluorometer (FLX800). After 90minutes the reactions were diluted with 1× sucrose loading buffer totwice the original reaction volume, and then 20 μl of this was rundirectly on a 2% agarose gel. Reactions either contained target DNA, orno target. We observed that appending cytosine residues to the primersresulted in faster amplification rate, while appending guanosinesresulted in defective amplification. Cytosine-appended primers alsoamplified ‘noise’ very rapidly.

FIG. 69 shows that appended 5′ sequences consisting of Thymine andCytosine residues demonstrate significant variation in amplificationbehaviour.

Primers were as indicated. J1 and K2 (SEQ IDs 66 and 69) are homologousto part of the Bacillus subtilis SpoOB locus. Reaction conditions were:

-   -   50 mM Tris pH 8.4    -   80 mM Potassium acetate    -   10 mM Magnesium acetate    -   2 mM DTT 5% PEG compound (Carbowax-20M)    -   3 mM ATP    -   30 mM Phosphocreatine    -   100 ng/μl creatine kinase    -   420 ng/μl gp32    -   140 ng/μl UvsX    -   35 ng/μl UvsY    -   200 μM dNTPs    -   300 nM each oligonucleotide    -   35 ng/μl Bsu polymerase    -   20 copies/μl B. subtilis genomic DNA    -   1:50,000 dilution from stock of SYBR green (Molecular probes)    -   Reaction volumes 50 μl, 37° C.

Amplification reactions were assessed in real-time employing SYBR greendye as described in FIG. 66. Conditions were similar, except that only20 copies/μl of start target template were employed, and the primersequences varied. Endpoint analysis was made by running productsgenerated at 60 minutes on a 2% agarose gel after dilution as describedfor FIG. 68. Reactions containing homopolymeric thymidine stretches, orvery thymidine-rich stretches, amplified somewhat poorly. Typically lessDNA was generated, and there was some indication that asymmetry ofamplification was occurring substantially. In general this data wasconsistent with cytosine residues being preferred in the 5′ sequences,and that other hard-to-determine phenomena were occurring, possibly someeffects of preferred recombinase ‘phasing’.

FIG. 70 shows more detailed study of the effects of 5′ appendedsequences.

We investigated yet more primers derived from the J1 and K2 primers (SEQIDs 66 and 69), mostly containing additional pyrimidine residues, butone pair containing a very 5′ guanosine residue. Some primers (6 and 12in this series) contained 5′ pyrimidines, but were shortened to a totalof 33 residues by removing bases at the 5′ (J1) or 3′ ends (K2)respectively. These primers thus shared less than 30 residues ofhomology with the genomic target. Primers were incubated under similarconditions to those used in FIGS. 69 and 70. The main differences werethat 630 ng/ml of gp32 were employed, and only 1 copy per microliter oftarget genomic DNA was used. Furthermore, in these experiments we alsoexamined the rate of amplification occurring in reactions containingonly single primers. In those reactions containing primer pairs, pairswere examined containing one oligonucleotide that is a J1 derivative,and one that is a K2 derivative. Pairs were used in which derivativeswere containing similar 5′ modifications. Notably single primeramplification rates were quite similar to two primer amplification ratesat this low target density, indicating that primer noise tends toinitiate as a competitive effect at about this very low target density.Oddly, primers 8 and 9, differing in only one cytosine residues, showedprofoundly different amplification behaviour. This may indicate a that aminimal stretch of cytosines is required for high rate activity, or thatsome specific phasing effect is in evidence.

FIG. 71 shows several strategies by which the specificity of sequencedetection might be improved by the use of ‘third probes’, and how theymight be configured to function with fluorophore/quencher pairs.

The presence of a specific DNA sequence can be detected by use of athird oligonucleotide probe which is blocked at the 3′ end, and hencedoes not engage in noisy amplification (at least without subsequentprocessing). Such third probes may form hybrids with the ampliconspecifically, and in such a duplex environment become the substrates forDNA processing enzymes which cleave the probe, thus separating thefluorophore and quencher. Given the nature of the recombinaseenvironment, fluorophore and quencher will be separated by no more thanabout 10-12 nucleotides. A selection of candidate nuclease is listed,and modified bases that would be included in the probes, or the backbonenature of the probe for each nuclease, are indicated.

FIG. 72 shows how a helix-distorting or base-specific nuclease can beemployed to detect specific sequenced including polymorphisms.

A single-stranded DNA is shown which has been coated with recombinase toform a homology-searching nucleoprotein filament. Arrows indicate theinteraction of such a structure will homologous duplexes, eitheridentical to the probe with regard to a single nucleotide polymorphism(SNP), or different (A instead of G). In both cases a strand exchangeoccurs successfully, and the outgoing strand is stabilised bysingle-stranded DNA binding protein. The resulting new duplexes areeither perfectly complementary, or contain a base mismatch, which willcause a local distortion of the helix. In the presence of a suitablestructure-specific nuclease the probe/template containing the distortionis specifically cleaved, either as nicks possibly on either strand, oras a double-strand break, depending on the nuclease. In other formats asimilar approach could be used to detect the presence or absence of aspecific sequence, regardless or not of polymorphic state, by includingmodified bases in the probe, such as 8-oxoguanine. In this case abase-removing and abasic-site-cleaving enzyme specific for 8-oxoguanineand duplex environments could be employed, such as E. coli Fpg protein(8-oxoguanine DNA glycosylase). In either case probe molecules mightcontain both a fluorophore and a quencher which are separated oncleavage.

FIG. 73 shows how dual-labeled fluorescent oligonucleotides showdifferential properties in an RPA environment compared to an environmentlacking saturating recombinase and single-stranded DNA binding proteins.

Upper left, three probes are shown which were used in an experiment toinvestigate the fluorescent properties of those oligonucleotides in thepresence or absence of proteins used in RPA environments. Conditionswere as follows:

-   -   50 mM Tris pH 8.4    -   80 mM Potassium acetate    -   10 mM Magnesium acetate    -   2 mM DTT    -   5% PEG compound (Carbowax-20M)    -   3 mM ATP    -   30 mM Phosphocreatine    -   100 ng/μl creatine kinase    -   200 μM dNTPs    -   120 nM each oligonucleotide as indicated    -   Optional:    -   630 ng/μl gp32    -   140 ng/μl UvsX    -   35 ng/μl UvsY

We observe that the FAM only containing oligonucleotide is slightlyquenched in the presence of DNA binding proteins, likely indicating somequenching of fluorescence by protein bound close to the fluorophore. Inmarked contrast, however, the oligonucleotides containing bothfluorophore and quencher show a reverse phenomenon, and this is markedlydifferent depending on whether they are 10 or 15 residues in length.More specifically both latter probes were significantly quenched in theabsence of proteins, consistent with the idea that formation of a randomcoil would ensure efficient quenching regardless of length. However inthe presence of proteins this quenching is reduced. In particular thequenching for the 15-mer is almost abolished, and it assumes afluorescence close to that level of the FAM only containingoligonucleotide in the presence of proteins. Conversely the 10-mer islittle-affected and remains highly quenched. This reduction in quenchingfor the 5-mer is most easily explained by the suggestion that in thepresence of DNA binding proteins the probe is stretched out into afairly rigid rod. For example UvsX and recA stretch single anddouble-stranded DNA to about 1.5 times the equivalent length of B-formDNA, and similar extension is described for gp32. Thus, and in contrastto free solution behaviour, fluorophore and quencher are highlyseparated in an RPA environment, even more so than in a DNA duplex, andare likely to become more quenched on hybridization (although notinvestigated in this experiment). A schematic is provided on theright-hand side indicating what the states of the 10-mer and 15-merprobe may be in the absence or presence of a recombinase environment,and potentially when hybridized to duplex DNA. Fluorophore and quencherare shown with a larger ‘excitation’ radius, which when overlappingwould lead to efficient fluorescence resonance energy transfer, and aconcomitant decrease in fluorescence emission.

FIG. 74 shows that an amplicon-probe containing a tetrahydrofuranylresidue becomes a substrate for the E. coli Nfo enzyme in an RPAamplification reaction, is cleaved, and that the cleavage product can beelongated.

RPA reactions were established using the B. subtilis genomic DNAspecific primers J1 and K2 (SEQ ID NO:66 and SEQ ID NO:69), the nestedprimer NEST26 (SEQ ID NO:77) and the biotin and Digoxygenin labeledprobe THF-Probe 1(5′-BIOTIN-CATGATTGGATGAATAAGCTGCAG-tetrahydrofuran-TGATTAAAGGAAAC-DIG-3′SEQ ID NO:80), which also contains a Tetrahydrofuranyl residue withinthe probe body. This probe is specific for the fragment amplified by J1and K2 primers, and overlaps the sequence of the NEST26 primer (allthese primers are discussed elsewhere). NEST26, a primer which generatesno noise on its own, was included so that a smaller nested product mightbe generated one of whose ends would be the target for the probe, justin case the probe was highly unstable in the topologically constrainedenvironment which will result from probe invasion into duplex J1/K2amplicon. Reactions had the following conditions:

-   -   50 mM Tris pH 7.9    -   80 mM Potassium acetate    -   10 mM Magnesium acetate    -   2 mM DTT    -   5% PEG compound (Carbowax-20M)    -   3 mM ATP    -   25 mM Phosphocreatine    -   100 ng/μl creatine kinase    -   600 ng/μl gp32    -   120 ng/μl UvsX    -   30 ng/μl UvsY    -   100 μM dNTPs    -   200 nM of J1 primer, K2 primer, and NEST26 primer    -   100 nM Probe    -   30 ng/μl Bsu polymerase    -   1000 copies/μl B. subtilis genomic DNA, or water controls as        indicated

Amplification reactions were phenol extracted, precipitated, andseparated on a 16.5% denaturing Urea-PAGE. Separated products weretransferred to nylon membrane and products were detected using aStreptavidin-HRP conjugate. As the 5′ end of the probe was biotinylated,this permitted the detection of uncut, cut and elongated probes. (A)Detection of free, and processed probes. Note that in the presence of anRPA environment capable of amplifying the target fragment, the probe waselongated preferentially elongated. The size of the elongated probe isconsistent with elongation to the end of the J1/K2 amplificationproduct, and this can occur because Nfo cleavage activity generates a3′-hydroxyl, thus ‘unblocking’ the probe and permitting syntheticextension. More elongation was seen when a higher concentration of Nfoenzyme was employed. In (B) the structure of the probe used in thisexperiment is shown. It comprises a sequence homologous to part of theamplicons generated by J1 and K2 primers, but contains atetrahydrofuranyl residue and is labeled at the 5′ end with biotin, andat the 3′ end (and thus blocked) with digoxygenin. In (C) a schematicrepresentation is given of the sequences of events thought to underlythe generation of elongated product via interaction of the probe withthe desired amplicon, cleavage and subsequent polymerase elongation ofthe free 3′ end. In this experiment apparent activity in reactionslacking start template is believed to arise from carry-overcontamination of the amplicon from previous laboratory experiments, aphenomenon that has become problematic in our laboratory due to the veryhigh sensitivity of RPA. Supporting evidence is provided in thesubsequent figure.

FIG. 75 shows that an amplicon-probe containing a tetrahydrofuranylresidue becomes a substrate for the E. coli Nfo enzyme in an RPAamplification reaction, is cleaved, and that the cleavage product can beelongated

In these experiments evidence is presented that the cleavage andextension of a tetrahydrofuranyl residue-containing amplicon-specificprobe is specific and dependant upon the presence of that targetsequences in the reaction, and also that having a free end overlappingthe probe target (to prevent topological strain of the probe/targetrecombination intermediate) is not necessary. In (A) the reaction setupis depicted schematically. The Bacillus subtilis locus targeted by theprimers J1, K2, L2, NEST26, (SEQ ID'S 66, 69, 71 AND 77) and the probeare depicted (the actual sequence is shown in FIG. 42B). Usually theprimers J1 and K2 are routinely used to amplify this locus and thus theprincipal laboratory contaminant is the fragment generated by these twoprimers. Thus we have also combined J1 with L2 in some experiments, thisfragment not being subject to amplification from carry-overcontamination from the former amplicon. In (B) are shown the results ofan experiment in which RPA reactions were configured to amplify eitherthe J1/K2 product, or the J1/L2 product, in the presence of added starttemplate (genomic DNA) or without. Included in the reaction are theprobe and the E. coli Nfo protein. In more detail the followingconditions were used:

-   -   50 mM Tris pH 7.9    -   80 mM Potassium acetate    -   10 mM Magnesium acetate    -   2 mM DTT    -   5% PEG compound (Carbowax-20M)    -   3 mM ATP    -   25 mM Phosphocreatine    -   100 ng/μl creatine kinase    -   600 ng/μl gp32    -   120 ng/μl UvsX    -   30 ng/μl UvsY    -   100 μM dNTPs    -   200 nM of J1 primer, 200 nM K2 OR L2 primer, and 200 nM NEST26        primer (lanes 1-4), in lanes 5 and 6 J1 and L2 primers were at        300 nM and NEST26 was not used 120 nM Probe    -   30 ng/μl Bsu polymerase    -   1000 copies/μl B. subtilis genomic DNA, or water controls as        indicated

Amplification reactions were phenol extracted, precipitated, andseparated on a 16.5% denaturing Urea-PAGE gel. Separated products weretransferred to nylon membrane and products were detected using aStreptavidin-HRP conjugate. As the 5′ end of the probe was biotinylated,this permitted the detection of uncut, cut and elongated probes. (A)Detection of probes, free and processed. Note that in the presence of anRPA environment capable of amplifying the target fragment, the probe waselongated preferentially elongated. Also note that while significantelongation occurs in the absence of the added target for the J1/K2primer pair (due we believe to contamination), when the J1/L2 primerspair is used this does not occur (a very faint band is, we believe, asample cross-loading artifact). Consequently we deduce that probecutting/elongation is a specific event. Furthermore the significantsignals in the no-template J1/K2 samples suggests that the system ishighly responsive to even very small numbers of targets, as ispresumably arising from the carry-over contamination. In (C) is shownthe results of a similar experiment in which the J1 and L2 primers wereused in combination with the probe on either no target DNA, or on asample with target DNA at 1000 copies per microliter (genomic DNA).Oligonucleotide concentrations are as follows: J1 and L2 at 240 nM,probe at just 12 nM, and ˜1000 ng per microliter of Nfo enzyme wereused. Other conditions are as described for the experiment in part (B).Samples were removed at the indicated times and stopped. Samples werephenol extracted, precipitated, and run on a denaturing gel as alreadydescribed, transferred to nylon membrane, and detected as describedabove. We note that cleavage/elongation products can be detected by 30minutes even when the probe is as low as 12 nM. This suggests that thekinetics of Nfo action under these conditions is sufficiently fast tomonitor reactions excellently in real-time.

FIG. 76 shows the general structure of several possible sorts of probethat might be used for real-time analysis of RPA reactions.

By combining the data suggesting that dual-labeled probes for use in RPAwould require relatively short separation distances to permit quenchingwith our knowledge of the efficient action of the E. coli Nfo protein onprocessing intermediates between THF-residue containing probes andduplex targets, we suggest several probe structures. In (A) and (B) weshow probes in which both a fluorophore and quencher are presentinternally (through base modifications), and between them is positioneda THF residue. The separation of the fluorophore and quencher is lessthan 10-12 residues to ensure efficient FRET between the groups. Theprobe is blocked at the 3′ end with a suitable group. Cleavage by theNfo enzyme at the THF residue will eliminate covalent association of thefluorophore and quencher, ultimately leading to an increase in solutionfluorescence. In (C) and (D) and alternative arrangement is depicted. Inthis case either fluorophore or quencher is positioned at the 3′terminusof the probe, thus blocking elongation. The other light-absorbing moietyis attached via and internal linkage slightly more 5′. The THF residueis positioned between these 2 groups, and the 2 light-absorbing groupsare separated by no more than 10-12 residues to ensure good FRET evenwhen stretched by DNA binding proteins.

DISCUSSION

There is a long-standing need for in vitro methods to amplify specificDNA sequences. Since the late 1980's the polymerase chain reaction (PCR)method has principally met this need (R. K. Saiki et al., Science 239,487-91 (Jan. 29, 1988)). The requirement for thermal cycling equipment,however, poses a significant barrier to the use of PCR outside of alaboratory setting. As described herein, we have developed a methodcalled RPA, which obviates the need for thermal melting of templateDNAs. RPA combines components of the bacteriophage T4recombination/replication system in vitro under critical definedconditions to mediate the hybridisation of oligonucleotide primers totemplate DNAs. Specifically, the bacteriophage T4 recombinase uvsX,single-stranded DNA binding (SSB) protein gp32, and recombinase loadingfactor uvsY together with molecular crowding agents, allow high fidelityin vitro recombinase-mediated DNA targeting. When this targeting systemis combined with strand displacement DNA synthesis mediated by enzymesof the E. coli or B. subtilis Pol I class, efficient exponential DNAamplification is achieved.

Any oligonucleotide sequence may be coated by recombinase to formhomology searching filaments (FIG. 37) giving RPA a broad utilityallowing amplification of virtually any DNA sequence. This feature hasbeen one of the major advantages of PCR over other in vitro DNAamplification methods (G. T. Walker, M. C. Little, J. G. Nadeau, D. D.Shank, Proc Natl Acad Sci USA 89, 392-6 (Jan. 1, 1992); D. Y. Zhang, M.Brandwein, T. Hsuih, H. B. L1, Mol Diagn 6, 141-50 (June, 2001); M.Vincent, Y. Xu, H. Kong, EMBO Rep 5, 795-800 (August, 2004); J. Compton,Nature 350, 91-2 (Mar. 7, 1991)). Resembling their in vivo role,homology-searching filaments scan duplex DNA for sequences complementaryto that of the oligonucleotide (T. Yonesaki, Y. Ryo, T. Minagawa, H.Takahashi, Eur J Biochem 148, 127-34 (Apr. 1, 1985); T. Shibata, C.DasGupta, R. P. Cunningham, C. M. Radding, Proc Natl Acad Sci USA 76,1638-42 (April, 1979)). On finding a match, the recombinase catalysesseveral reactions: the primer is paired with its complement, the similar‘outgoing’ strand is displaced, and the recombinase dissociates. Thisestablishes a ‘D-loop’ structure accessible to other reactioncomponents. Exchange events occurring away from a free DNA end generatetopologically strained joints, as the outgoing strand is attached onboth sides of the exchanged region (FIG. 37C).

Embedded sequences generate topologically constrained intermediates thatare unstable. Joints formed at DNA ends permit free rotation of thedisplaced strand (P. W. Riddles, I. R. Lehman, J Biol Chem 260, 165-9(Jan. 10, 1985)). Because these two structures have differentstabilities elongation of initial strand invasion events are lessefficient than subsequent ones (FIG. 39B). The free 3′ end of theoligonucleotide primes synthesis by a strand-displacing DNA polymerasesuch as the Klenow fragment of E. coli, or the Bacillus subtilis DNApolymerase I (Bsu). The synthetic and strand-displacing activities ofthe polymerase result in the production of a double-stranded DNA and adisplaced single strand. This displaced strand is replicated either bydirect hybridisation and elongation of the second oligonucleotide, or bystrand displacement synthesis if an invasion event had already occurredfrom the opposite end. The generation of two complete daughter duplexescompletes one round of RPA. Invasions continue to act on products ofprevious synthesis reactions with the end targeted products eventuallydominating the reaction.

In developing the method of the invention, we have found that severalimportant conditions are important for optimal RPA to occur. First,there needs to be saturating quantities of nucleic acid melting proteinspresent in the reaction especially a SSB such as gp32. Second, thereneeds to be a sufficient quantity of recombinase-loaded primer toachieve an acceptable invasion/strand-exchange rate. Finally, therecombinase/single-stranded DNA primer filaments need to be dynamic andcapable of disassembly. There are competing biochemical activities ofthe reaction components. For example, in a typical in vitro situationrecombinases are usually out-competed by saturating amounts of SSBs suchas gp32.

To overcome this problem, others have used nonhydrolysable ATP analoguessuch as ATP-γ-S, which stabilises the recombinase/single-stranded primerDNA interaction (S. C. Kowalczykowski, J. Clow, R. Somani, A. Varghese,J Mol Biol 193, 81-95 (Jan. 5, 1987); A. L. Eggler, S. L. Lusetti, M. M.Cox, J Biol Chem 278, 16389-96 (May 2, 2003); T. Shibata, C. DasGupta,R. P. Cunningham, C. M. Radding, Proc Natl Acad Sci USA 77, 2606-10(May, 1980)). Non-hydrolysable ATP analogues are, however, incompatiblewith the dynamic activity of recombinase/single-stranded DNA primerfilaments needed to complete strand-exchange reactions and allowpolymerases access to the D-loops (L. Xu, K. J. Marians, J Biol Chem277, 14321-8 (Apr. 19, 2002); P. W. Riddles, I. R. Lehman, J Biol Chem260, 170-3 (Jan. 10, 1985); N. Armes, D. Stemple, in patent applicationPCT WO 03/072805 (ASM Scientific, Inc., USA, 2003)) (FIG. 38).

There are other ways the recombinase/single-stranded primer DNAinteraction could be stabilised. For example, another bacteriophage T4protein called uvsY is known to aid loading of uvsX onto gp32-coated DNA(L. D. Harris, J. D. Griffith, J Mol Biol 206, 19-27 (Mar. 5, 1989)). Inaddition, molecular crowding agents are also known to facilitate loadingand stabilisation of the E. coli recA recombinase protein (P. E. Layery,S. C. Kowalczykowski, J Biol Chem 267, 9307-14 (May 5, 1992)). Wetherefore tested whether uvsY and molecular crowding agents mightalleviate the unfavourable competition between uvsX and gp32 in adynamic, ATP-dependent system.

Titration of reaction components reveals that defined quantities of T4gp32, T4 uvsX, T4 uvsY, ATP, and PEG are required for DNA amplification(FIG. 38). Indeed the T4 uvsY recombinase mediator protein andPEG-compound (Carbowax 20M) are required to achieve detectableamplification. At reduced concentrations of gp32, amplificationefficiency is impaired generating smearing and laddering of reactionproducts. The recombinase uvsX protein is important for RPA and thereaction rate is accelerated at higher concentrations, although this canalso increase artifacts (FIG. 39A). ATP is important for the reaction,and we have found that an ATP regeneration system is needed to reachdetectable levels of product for most reactions. Conversely, ATP-γ-S isa powerful inhibitor of amplification.

To be a useful tool for routine DNA amplification applications such asdiagnostic testing, RPA should be sensitive and specific, applicable todiverse sequence targets and be capable of amplifying fragments ofsufficient size. We first investigated the size of products that couldbe generated. Amplified products up to 1000 base pairs in size could beamplified using standard reaction conditions (FIG. 38A). Largeramplified products may also be generated using RPA.

We tested the sensitivity of RPA under the most stringent conditions,using a single-step RPA reaction, without nesting, and detectingproducts by conventional ethidium bromide staining of agarose gels. Withseveral independent primer/target sets we routinely detected less than10 copies of starting duplex template. We observed variability betweenexperiments at lowest detectable copy number but all attempts weresuccessful at detecting less than 10 copies. With proper samplehandling, RPA may be used to amplify from single molecules to detectablelevels in a single reaction. To explore this possibility, we amplified apolymorphic simple tandem repeat (STR) marker from human DNA diluteduntil only a few copies should remain. We generated a number ofamplified products corresponding to both possible separate allelespresent in the sample DNA (FIG. 39E,F). This allele separation effectsuggests that RPA has single molecule sensitivity and thus surpasses thesensitivity of many other DNA amplification methods.

Analysis of the amount of amplified product shows that RPA willroutinely amplify DNA samples by 10¹¹⁻¹²-fold from small quantities ofstarting template. Final product levels are typically in the range of10-250 nM, generating more than sufficient quantities of DNA for eventhe least sensitive detection protocols. To assess specificity ofamplification reactions we have analysed many primer pairs, mostdirected to human DNA sequences. For every primer/template set, we havetested the predicted product sizes, restriction enzyme digestionpatterns, or product DNA sequence to show that amplification isspecific. We have not observed amplification of non-target sequencesfrom sample DNA to the best interpretation of our data. Artifacts wehave observed are product or primer related (FIG. 39A).

Often in diagnostic settings, specificity problems arise from the largeamounts of nontarget DNA present in a sample. For example, in pathogendetection, human DNA from blood samples can interfere with detection ofpathogen DNA. We therefore sought to detect trace quantities of targetDNA in a large mass of unrelated DNA. We found that RPA was able toamplify a target to detectable levels from 100 copies of Bacillussubtilis DNA in the presence of 1 μg of human DNA (i.e., 10⁸-fold lessB. subtilis DNA than human DNA by mass). With such a large mass ofsample DNA we found we had to increase levels of uvsX and uvsY comparedto equivalent reactions without excess competitor DNA to achieve anacceptable reaction rate. This is perhaps due to out-titration of thehomology-searching components.

In time-course experiments with several different primer/template sets,we found that amplification rate was partially dependent on productlength. For fragments in the range 150-400 base pairs, however, fairlysimilar rates were observed, allowing amplification from hundreds tothousands of starting copies to gel detectable levels (˜10¹² copies) inas little as 20 minutes). We estimate that we have been able to reduceaverage ‘cycle’ times to as little as 30 seconds on average for suchfragments. Using optimally short target sequences and sensitivedetection method, we expect that a diagnostic amplification/detectionassay could be performed well within an hour. Our studies show that fora large number of arbitrary DNA targets in complex samples high-qualityprimer pairs can be easily designed. We have addressed minimaloligonucleotide length and found that while oligonucleotides of lessthan 30 nucleotides do not amplify DNA effectively, those of 30-35nucleotides in length are excellent primers and are short enough foreasy synthesis (FIG. 40).

As demonstrated herein, RPA is an excellent general method to amplifyspecific DNA sequences. We have shown that RPA reactions can bemonitored in real-time using minor groove binding dyes and demonstratean excellent capacity to assess start target copy number. Furthermoremultiple targets can be co-amplified, and sequence-specific real-timesensors (‘third probes’) can be readily included in the reactionenvironment. We have identified and demonstrated that DNA repair enzymessuch as fpg, Nth and particularly Nfo operate in RPA reactions and canbe combined with suitable probes to permit real-time assessment ofreaction behaviour in a sequence-specific manner. We show thatfluorescence-based probe systems show markedly different properties inthe RPA environment to conventional PCR reaction environments, orsimilar. Finally our initial experiments indicate that it is easy tolyophilise the components of the RPA reaction for convenient storage andreconstitution (FIG. 40). This method, which operates robustly atconstant low temperature, can be lyophilised for easy storage andrequires no thermal cycling or melting for high sensitivity, nor othercomplex handling, and can be readily monitored in real-time, offers asignificant breakthrough in the development of DNA diagnostic, forensicand other point-of-use applications. Once integrated with portablesample DNA extraction and product detection systems, RPA should enableeasy-to-use clinical or domestic testing kits for a variety of pathogens(e.g., Clamydia or MRSA) as well as field kits for other applications.

The details of one or more embodiments of the invention have been setforth in the accompanying description above. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. Other features, objects, and advantagesof the invention will be apparent from the description and from theclaims.

In the specification and the appended claims, the singular forms includeplural referents unless the context clearly dictates otherwise. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs. Unless expressly stated otherwise,the techniques employed or contemplated herein are standardmethodologies well known to one of ordinary skill in the art. Allpatents, patent applications and publications cited in thisspecification are hereby incorporated by reference herein.

1-222. (canceled)
 223. A composition comprising; a T4 bacteriophageUvsX; a T4 bacteriophage single-stranded DNA binding protein (gp32); anda DNA polymerase.
 224. The composition of claim 223, wherein the DNApolymerase is selected from the group consisting of E. coli DNApolymerase I Klenow fragment, B. stearothermophilus polymerase (Bst), B.subtilis Phi-29 polymerase, and B. subtilis polymerase I (Bsu).
 225. Thecomposition of claims 223 or 224, further comprising a crowding agent,ATP or an ATP analog, dNTP(s), or T4 bacteriophage UvsY.
 226. Thecomposition of claim 225, wherein the crowding agent is selected fromthe group consisting of polyethylene glycol (PEG), dextran and Ficoll.227. The composition of claim 22, wherein the PEG is PEG1450, PEG3000,PEG8000 or PEG10000.
 228. The composition of claim 226, wherein the PEGhas a molecular weight between 15000 and
 20000. 229. The composition ofclaim 225, wherein the dNTP(s) is/are selected from the group consistingof dATP, dGTP, dCTP and dTTP.
 230. The composition of claim 225, whereinthe ATP or ATP analog is selected from the group consisting of ATP,ATP-γ-S, ATB-β-S, ddATP, and a combination thereof.